Compositions and methods involving respiratory syncytial virus subgroup B strain 9320

ABSTRACT

The complete polynucleotide sequence of the human respiratory syncytial virus subgroup B strain 9320 genome is provided. Proteins encoded by this polynucleotide sequence are also provided. Isolated or recombinant RSV (e.g., attenuated recombinant RSV), nucleic acids, and polypeptides, e.g., comprising mutations in the attachment protein G, are also provided, as are immunogenic compositions comprising such isolated or recombinant RSV, nucleic acids, and polypeptides. Related methods are also described.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application is a non-provisional utility patent applicationclaiming priority to and benefit of the following prior provisionalpatent applications: U.S. Ser. No. 60/458,331, filed Mar. 28, 2003,entitled “Compositions and Methods Involving Respiratory Syncytial VirusSubgroup B Strain 9320” by Xing Cheng, et al., and U.S. Ser. No.60/508,320, filed Oct. 3, 2003, entitled “Compositions and MethodsInvolving Respiratory Syncytial Virus Subgroup B Strain 9320” by XingCheng, et al., each of which is incorporated herein by reference in itsentirety for all purposes.

FIELD OF THE INVENTION

[0002] The present invention is in the field of virology. Morespecifically, the invention relates to human respiratory syncytialvirus, including the diagnosis, treatment, and prevention of human RSVinfections.

BACKGROUND OF THE INVENTION

[0003] Human Respiratory Syncytial Virus (RSV) is the leading cause ofhospitalization for viral respiratory tract disease (e.g., bronchiolitisand pneumonia) in infants and young children worldwide, as well as asignificant source of morbidity and mortality in immunocompromisedadults and in the elderly (see, e.g., Shay et al. (1999)“Bronchiolitis-associated hospitalizations among U.S. children,1980-1996” JAMA 282:1440-1446, Falsey et al. (1995) “Respiratorysyncytial virus and influenza A infections in the hospitalized elderly”J Infect Dis 172:389-394, Falsey et al. (1992) “Viral respiratoryinfections in the institutionalized elderly: clinical and epidemiologicfindings” J Am Geriatr Soc 40:115-119, Falsey and Walsh (1998)“Relationship of serum antibody to risk of respiratory syncytial virusinfection in elderly adults” J Infect Dis 177:463-466, Hall et al.(1986) “Respiratory syncytial viral infection in children withcompromised immune function” N Engl J Med 315:77-81, and Harrington etal. (1992) “An outbreak of respiratory syncytial virus in a bone marrowtransplant center” J Infect Dis 165:987-993). To date, no vaccines havebeen approved which are able to prevent the diseases associated with RSVinfection. RSV is an enveloped virus that has a single-stranded negativesense non-segmented RNA genome, and it is classified in the Pneumovirusgenus of the Paramyxoviridae family (Collins et al. (2001) Respiratorysyncytial virus. pp. 1443-1485. In; Knipe and Howley (eds.) FieldsVirology vol. 1. Lippincott, Williams and Wilkins, Philadelphia; Lamband Kolakofsky (2001) Paramyxoviridae: the viruses and their replicationpp. 1305-1340. In; Knipe and Howley (eds.) Fields Virology vol. 1.Lippincott, Williams and Wilkins, Philadelphia). Human RSV is classifiedinto two subgroups, subgroups A and B, based on antigenic diversity andnucleotide sequence divergence. For example, the attachment protein G ismost divergent and the fusion protein F is relatively conserved betweenthe two subgroups.

[0004] Considerable progress has been made towards understanding themolecular biology of subgroup A RSV; however, much less information isavailable for subgroup B RSV. Most work to date has focused on subgroupA strains. For example, RSV strain A2 has been sequenced. The genome ofthe A2 strain RSV is 15,222 nt in length and contains 10 transcriptionalunits that encode 11 proteins (NS1, NS2, N, P, M, SH, G, F, M2-1, M2-2,and L). The genome is tightly bound by the N protein to form thenucleocapsid, which is the template for the viral RNA polymerasecomprising the N, P and L proteins (Grosfeld et al. (1995) J. Virol.69:5677-5686; Yu et al. (1995) J. Virol. 69:2412-2419). Eachtranscription unit is flanked by a highly conserved 10-nt gene-start(GS) signal, at which mRNA synthesis begins, and ends with asemiconserved 12- to 13-nt gene-end (GE) signal that directspolyadenylation and release of mRNAs (Harmon et al. (2001) J. Virol.75:36-44; Kuo et al. (1996) J. Virol. 70:6892-6901). Transcription ofRSV genes is sequential, and there is a gradient of decreasing mRNAsynthesis due to transcription attenuation (Barik (1992) J. Virol.66:6813-6818; Dickens et al. (1984) J. Virol. 52:364-369). The viral RNApolymerase must first terminate synthesis of the upstream message inorder to initiate synthesis of the downstream mRNA.

[0005] The nucleocapsid protein (N), phosphoprotein (P), and largepolymerase protein (L) constitute the minimal components for viral RNAreplication and transcription in vitro (Grosfield et al. (1995) J.Virol. 69:5677-5686; Yu et al. (1995) J. Virol. 69:2412-2419). The Nprotein associates with the genomic RNA to form the nucleocapsid, whichserves as the template for RNA synthesis. The L protein is amultifunctional protein that contains RNA-dependent RNA polymerasecatalytic motifs and is also probably responsible for capping andpolyadenylation of viral mRNAs. However, the L protein alone is notsufficient for the polymerase function; the P protein is also required.Transcription and replication of RSV RNA are also modulated by the M2-1,M2-2, NS1, and NS2 proteins that are unique to the pneumoviruses. M2-1is a transcription antitermination (or elongation) factor required forprocessive RNA synthesis and transcription read-through at genejunctions, essential for RNA transcription and virus replication(Collins et al. (1996) “Transcription elongation factor of respiratorysyncytial virus, a nonsegmented negative-strand RNA virus” Proc NatlAcad Sci USA 93:81-85; Hardy and Wertz (2000) “The Cys3-His1 motif ofthe respiratory syncytial virus M2-1 protein is essential for proteinfunction” J Virol 74:5880-5885; Tang et al. (2001) “Requirement ofcysteines and length of the human respiratory syncytial virus M2-1protein for protein function and virus viability” J Virol75:11328-11335; Collins et al. (2001) in D. M. Knipe et al. (eds.),Fields Virology, 4^(th) ed. Lippincott, Philadelphia; Hardy et al.(1999) J. Virol. 73:170-176; and Hardy andWertz (1998) J. Virol.72:520-526). M2-2, though not essential for virus replication in tissueculture, is involved in the switch between viral RNA transcription andreplication (Bermingham and Collins (1999) Proc. Natl. Acad. Sci. USA96:11259-11264; Jin et al. (2000) J. Virol. 74:74-82). NS1 and NS2 havebeen shown to inhibit minigenome synthesis in vitro (Atreya et al.(1998) J. Virol. 72:1452-1461).

[0006] NS1, NS2, SH, M2-2 and G are accessory proteins that can bedeleted from the RSV A2 strain without affecting virus viability(Bermingham and Collins (1999) Proc. Natl. Acad. Sci. USA96:11259-11264; Jin et al. (2000) J. Virol. 74:74-82; Jin et al. (2000)“Recombinant respiratory syncytial viruses with deletions in the NS1,NS2, SH, and M2-2 genes are attenuated in vitro and in vivo” Virology273:210-218; Bukreyev et al. (1997) “Recombinant respiratory syncytialvirus from which the entire SH gene has been deleted grows efficientlyin cell culture and exhibits site- specific attenuation in therespiratory tract of the mouse” J Virol 71:8973-8982; Teng and Collins(1999) “Altered growth characteristics of recombinant respiratorysyncytial viruses which do not produce NS2 protein” J Virol 73:466-473;Teng et al. (2000) “Recombinant respiratory syncytial virus that doesnot express the NS1 or M2-2 protein is highly attenuated and immunogenicin chimpanzees” J Virol 74:9317-9321; Karron et al. (1997) “Respiratorysyncytial virus (RSV) SH and G proteins are not essential for viralreplication in vitro: clinical evaluation and molecular characterizationof a cold-passaged, attenuated RSV subgroup B mutant” Proc Natl Acad SciUSA 94:13961-13966). However, except for the SH deletion mutant, most ofthe gene deletion mutants do not replicate as well as the wild type RSVeither in tissue culture or in animal hosts.

[0007] The G and F proteins are the two major surface antigens thatelicit anti-RSV neutralizing antibodies to provide protective immunityagainst RSV infection and reinfection. High levels of circulatingantibodies correlate with protection against RSV infections or reductionof disease severity (Crowe (1999) Microbiol. Immunol. 236:191-214). Asnoted, two antigenic RSV subgroups (A and B) have been recognized basedon virus antigenic and sequence divergence (Anderson et al. (1985) J.Infect. Dis. 151:626-633; Mufson et al. (1985) J. Gen. Virol.66:2111-2124). By using a reciprocal cross-neutralization assay, it hasbeen determined that the F proteins between the two subgroups are 50%related and the G proteins are only 1-7% related (reviewed by Collins etal. (2001) “Respiratory syncytial virus” In: D. M. Knipe et al. (Ed)Fields Virology, pp. 1443-1485, Vol. 1, Lippincott Williams & Wilkins,Philadelphia). This antigenic diversity may be partly responsible forrepeated RSV infection. The antigenic diversity of these two RSVsubgroups enables viruses from both subgroups to circulate concurrentlyin a community, and the prevalence of each subgroup can alternate duringsuccessive years. Epidemic studies of RSV infection in children havesuggested that naturally acquired infection elicits a relatively higherprotection against disease caused by the homologous subgroup virus(McIntosh and Chanock (1990) “Respiratory syncytial virus” In: D. M.Knipe et al. (Ed) Second Edition Virology, pp.1045-1072, Raven Press,Ltd., New York). The immunity induced by RSV infection is transient andsubsequent reinfection can occur. However, RSV reinfection usually doesnot cause severe disease. An RSV vaccine is therefore typically targetedto provide protection against severe lower respiratory disease caused byRSV subgroup A and B viruses.

[0008] Efforts to produce a safe and effective RSV vaccine have focusedon the administration of purified viral antigen or the development oflive attenuated RSV for intranasal administration. For example, aformalin-inactivated virus vaccine not only failed to provide protectionagainst RSV infection, but was shown to exacerbate symptoms duringsubsequent infection by the wild-type virus in infants (Kapikian et al.(1969) Am. J. Epidemiol. 89:405-421; Chin et al. (1969) Am. J.Epidemiol. 89:449-63). More recently, efforts have been aimed towardsdeveloping live attenuated temperature-sensitive mutants by chemicalmutagenesis or cold passage of the wild-type RSV (Crowe et al. (1994)Vaccine 12:691-9). Typically, the virus candidates have been eitherunderattenuated or overattenuated (Kim et al. (1973) Pediatrics52:56-63; Wright et al. (1976) J. Pediatrics 88:931-6), and some of thecandidates were genetically unstable, resulting in the loss of theattenuated phenotype (Hodges et al. (1974) Proc Soc. Exp. Bio. Med.145:1158-64). To date, no live attenuated vaccine has been brought tomarket.

[0009] Characterization of additional strains of RSV, particularly fromsubgroup B, will assist in production of effective vaccines (e.g.,regions of homology or identity between strains can indicatefunctionally conserved regions that can be targeted by mutagenesis).Although short regions of various subgroup B strains have been sequenced(e.g., B9320 protein G, SEQ ID NO: 14, from GenBank accession numberM73544; a B9320 intergenic region, SEQ ID NO: 15, from GenBank accessionnumber S75820; B9320 G and F gene start and end sequences, SEQ IDNOs:16-19, from Jin et al. (1998) Virology 251:206-214 and Cheng et al.(2001) Virology 283:59-68; and various B18537 coding and intergenicregions, GenBank accession numbers D00334, D00392-D00397, D00736,D01042, and M17213), only one subgroup B strain, strain B1, has beensequenced in its entirety (SEQ ID NO: 13, from GenBank accession numberAF013254).

[0010] Accordingly, this invention presents the complete polynucleotidesequence of human RSV subgroup B strain 9320. Polypeptides encoded bythe B9320 genome are also provided, as are other benefits which willbecome apparent upon review of the disclosure.

SUMMARY OF THE INVENTION

[0011] The present invention provides the complete polynucleotidesequence of human respiratory syncytial virus subgroup B strain 9320.Amino acid sequences of proteins encoded by the B9320 genome are alsoprovided. The invention provides isolated or recombinant nucleic acidsand polypeptides comprising the novel B9320 sequences. Isolated orrecombinant RSV comprising the nucleic acids and polypeptides of theinvention (e.g., attenuated recombinant RSV) are also provided, as areimmunogenic compositions including such nucleic acids, polypeptides, andRSV that are suitable for use as vaccines. Recovery of infectiousrecombinant 9320 viruses from cDNAs is described.

[0012] In a first aspect, the present invention provides isolated orrecombinant nucleic acids comprising a polynucleotide sequence of theinvention. Thus, for example, an isolated or recombinant nucleic acidcomprising the polynucleotide sequence of SEQ ID NO:1 or a complementarypolynucleotide sequence thereof is a favored embodiment of theinvention. An isolated or recombinant nucleic acid comprising at leastone unique polynucleotide subsequence of SEQ ID NO:1 or a complementarypolynucleotide sequence thereof, with the proviso that the uniquepolynucleotide subsequence includes at least one subsequence notincluded in SEQ ID NOs:14-19 or a complementary polynucleotide sequencethereof, is another favored embodiment. The unique polynucleotidesubsequence can, for example, comprise at least 10 contiguousnucleotides of SEQ ID NO:1 or a complementary polynucleotide sequencethereof (e.g., at least 20 contiguous nucleotides, at least 50contiguous nucleotides, at least 100 contiguous nucleotides, at least500 contiguous nucleotides, or even at least 1000 contiguousnucleotides). In some embodiments, the polynucleotide subsequenceincludes at least one compete open reading frame (ORF) of SEQ ID NO:1.

[0013] In addition to the sequences explicitly provided in theaccompanying sequence listing, polynucleotide sequences that are highlyrelated structurally and/or functionally (e.g., as defined byhybridization and/or sequence identity) are polynucleotides of theinvention. For example, a polynucleotide sequence that is greater than97.8% identical to SEQ ID NO:1 or a complementary polynucleotidesequence thereof, as determined by BLASTN using default parameters, is apolynucleotide of the invention. As another example, a polynucleotidesequence that hybridizes under stringent conditions over substantiallythe entire length of a polynucleotide subsequence comprising at least100 contiguous nucleotides of SEQ ID NO:1 or a complementarypolynucleotide sequence thereof, wherein the polynucleotide sequencehybridizes to the polynucleotide subsequence of SEQ ID NO:1 or thecomplementary polynucleotide sequence thereof under said stringentconditions with at least 2× a signal to noise ratio that thepolynucleotide sequence hybridizes to a corresponding polynucleotidesubsequence of SEQ ID NO:13 or a complementary polynucleotide sequencethereof, is a polynucleotide sequence of the invention. Similarly,polynucleotide sequences of the invention include a polynucleotidesequence encoding a polypeptide of the invention, e.g., encoding anamino acid sequence or unique subsequence selected from the groupconsisting of SEQ ID NOs:2-11 or an artificial conservative variationthereof.

[0014] A nucleic acid of the invention optionally comprises at least oneartificially mutated nucleotide, e.g., at least one artificiallydeleted, inserted, and/or substituted nucleotide. In certainembodiments, mutation of the polynucleotide sequence results inalteration of an encoded amino acid sequence. Thus, in one class ofembodiments, at least one polypeptide encoded by the nucleic acidcomprises at least one deleted, inserted, and/or substituted amino acidresidue (e.g., at least one conservatively or non-conservativelysubstituted amino acid residue). For example, the mutated nucleotide canbe located in an ORF encoding a polypeptide selected from SEQ IDNOs:2-12.

[0015] Another class of embodiments provides vectors comprising thenucleic acids of the invention. Yet another class of embodimentsprovides a host cell into which such a vector has been introduced.Another class of embodiments provides methods of producing a recombinantrespiratory syncytial virus. In the methods, such a host cell iscultured in a suitable culture medium under conditions permittingexpression of the nucleic acid, and the recombinant respiratorysyncytial virus is isolated from the host cell and/or the medium.Recombinant RSV produced according to these methods form another featureof the invention, as do recombinant RSV comprising a nucleic acid of theinvention. A related class of embodiments provides methods of producingan isolated or recombinant polypeptide. In the methods, a host cellcomprising a vector that includes a nucleic acid of the invention iscultured in a suitable culture medium under conditions permittingexpression of the nucleic acid, and the polypeptide is isolated from thehost cell and/or the medium. Polypeptides produced according to thesemethods form another feature of the invention, as do polypeptidescomprising an amino acid sequence or subsequence that is encoded by anucleic acid of the invention.

[0016] One aspect of the invention provides isolated or recombinantpolypeptides comprising an amino acid sequence of the invention. Thus,for example, an isolated or recombinant polypeptide comprising an aminoacid sequence selected from the group consisting of SEQ ID NOs:2-11 is afavored embodiment of the invention. An isolated or recombinantpolypeptide comprising a unique amino acid subsequence comprising atleast 7 (e.g., at least 8, at least 10, at least 20, at least 50, ormore) contiguous amino acid residues of any one of SEQ ID NOs:2-11 isanother favored embodiment. Artificial conservative variations of aminoacid sequences or subsequences of the invention are also amino acidsequences of the invention, as are amino acid sequences that aresubstantially identical to an amino acid sequence of the invention. Forexample, an amino acid sequence that is greater than 99.3% identical toSEQ ID NO:2, greater than 98.4% identical to SEQ ID NO:3, greater than99.7% identical to SEQ ID NO:4, greater than 98.3% identical to SEQ IDNO:5, greater than 99.6% identical to SEQ ID NO:6, greater than 97.0%identical to SEQ ID NO:7, greater than 99.3% identical to SEQ ID NO:8,greater than 99.5% identical to SEQ ID NO:9, greater than 96.4%identical to SEQ ID NO:10, or greater than 99.2% identical to SEQ IDNO:11, as determined by BLASTP using default parameters, is an aminoacid sequence of the invention. Similarly, an amino acid sequence orsubsequence that is specifically bound by an antibody that specificallybinds to an amino acid sequence or subsequence encoded by SEQ ID NO:1,wherein said antibody does not specifically bind to an amino acidsequence or subsequence encoded by SEQ ID NO:13 or SEQ ID NO:14, is anamino acid sequence of the invention.

[0017] A polypeptide of the invention optionally comprises at least oneartificially altered amino acid, e.g., at least one deleted, inserted,and/or substituted amino acid. For example, one class of embodimentsprovides an isolated or recombinant polypeptide comprising the aminoacid sequence of SEQ ID NO:12 with a deletion of residues 164-197, or anartificial conservative variation thereof.

[0018] Immunogenic compositions comprising an immunologically effectiveamount of a recombinant respiratory syncytial virus, polypeptide, and/ornucleic acid of the invention form another aspect of the invention.Similarly, another feature of the invention provides methods forstimulating the immune system of an individual to produce a protectiveimmune response against respiratory syncytial virus. In the methods, animmunologically effective amount of a respiratory syncytial virus,polypeptide, and/or nucleic acid of the invention is administered to theindividual in a physiologically acceptable carrier.

BRIEF DESCRIPTION OF THE DRAWINGS

[0019]FIG. 1 schematically illustrates assembly of the full-lengthantigenomic RSV 9320 cDNA. Positions of various subclones used toassemble the full length antigenomic cDNA are indicated. The cDNAfragments obtained by RT/PCR were ligated through the indicatedrestriction enzyme sites. The fourth residue of the leader sequence atthe antigenomic sense was either C or G as indicated.

[0020]FIG. 2 schematically illustrates the cloning of pB-L, the assemblyof the L coding region from three subclones. Positions of varioussubclones and primers used to assemble pB-L are indicated.

[0021]FIG. 3 schematically illustrates cloning of the 5′ portion of theRSV 9320 antigenome. Positions of various subclones and primers areindicated.

[0022]FIG. 4 presents line graphs illustrating the growth of wild type9320 (diamonds), rg9320C4 (squares), rg9320G4(triangles), and rg9320ΔG(circles) in Vero cells (Panel A) and HEp-2 cells (Panel B). Vero orBEp-2 cells were infected with each virus at an m.o.i of 0.1 andincubated at 35° C. for 5 days. The viruses released into the culturesupernatants at each day were titrated in Vero cells by plaque assay.

DEFINITIONS

[0023] Unless defined otherwise, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which the invention pertains. The followingdefinitions supplement those in the art and are directed to the currentapplication and are not to be imputed to any related or unrelated case,e.g., to any commonly owned patent or application. Although any methodsand materials similar or equivalent to those described herein can beused in the practice for testing of the present invention, the preferredmaterials and methods are described herein. Accordingly, the terminologyused herein is for the purpose of describing particular embodimentsonly, and is not intended to be limiting.

[0024] As used in this specification and the appended claims, thesingular forms “a,” “an” and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “avirus” includes a plurality of viruses; reference to “a host cell”includes mixtures of host cells, and the like.

[0025] An “amino acid sequence” is a polymer of amino acid residues (aprotein, polypeptide, etc.) or a character string representing an aminoacid polymer, depending on context.

[0026] A “polynucleotide sequence” or “nucleotide sequence” is a polymerof nucleotides (an oligonucleotide, a DNA, a nucleic acid, etc.) or acharacter string representing a nucleotide polymer, depending oncontext. From any specified polynucleotide sequence, either the givennucleic acid or the complementary polynucleotide sequence (e.g., thecomplementary nucleic acid) can be determined.

[0027] A “subsequence” is any portion of an entire sequence, up to andincluding the complete sequence. Typically, a subsequence comprises lessthan the full-length sequence. A “unique subsequence” is a subsequencethat is not found in any previously determined RSV polynucleotide orpolypeptide sequence (e.g., the A2, B1, and B18537 sequences listedand/or referenced herein).

[0028] An “artificial mutation” is a mutation introduced by humanintervention, e.g., under laboratory conditions. Thus, an “artificiallymutated” nucleotide is a nucleotide that has been mutated as a result ofhuman intervention, an “artificially altered” amino acid residue is aresidue that has been altered as a result of human intervention, and an“artificial conservative variation” is a conservative variation that hasbeen produced by human intervention. For example, a wild type virus(e.g., one circulating naturally among human hosts) or other parentalstrain of virus can be “artificially mutated” using recombinant DNAtechniques to alter the viral genome (e.g., the viral genome can bealtered by in vitro mutagenesis, or by exposing it to a chemical,ionizing radiation, or the like and then performing in vitro or in vivoselection for a temperature sensitive, cold sensitive, or otherwiseattenuated strain of virus). As another example, a wild type protein canbe “artificially altered” by artificially mutating the gene encodingthat protein.

[0029] The term “variant” with respect to a polypeptide refers to anamino acid sequence that is altered by one or more amino acids withrespect to a reference sequence. The variant can have “conservative”changes, wherein a substituted amino acid has similar structural orchemical properties, e.g., replacement of leucine with isoleucine.Alternatively, a variant can have “nonconservative” changes, e.g.,replacement of a glycine with a tryptophan. Analogous minor variationcan also include amino acid deletion or insertion, or both. Guidance indetermining which amino acid residues can be substituted, inserted, ordeleted without eliminating biological or immunological activity can befound using computer programs well known in the art, for example,DNASTAR software. Examples of conservative substitutions are alsodescribed below.

[0030] The term “nucleic acid” or “polynucleotide” encompasses anyphysical string of monomer units that can be corresponded to a string ofnucleotides, including a polymer of nucleotides (e.g., a typical DNA orRNA polymer), PNAs, modified oligonucleotides (e.g., oligonucleotidescomprising bases that are not typical to biological RNA or DNA insolution, such as 2′-O-methylated oligonucleotides), and the like. Anucleic acid can be e.g., single-stranded or double-stranded. Unlessotherwise indicated, a particular nucleic acid sequence of thisinvention encompasses complementary sequences, in addition to thesequence explicitly indicated.

[0031] The term “gene” is used broadly to refer to any nucleic acidassociated with a biological function. Thus, genes include codingsequences and/or the regulatory sequences required for their expression.The term “gene” applies to a specific genomic sequence, as well as to acDNA or an mRNA encoded by that genomic sequence. Genes also includenon-expressed nucleic acid segments that, for example, form recognitionsequences for other proteins. Non-expressed regulatory sequences include“promoters” and “enhancers,” to which regulatory proteins such astranscription factors bind, resulting in transcription of adjacent ornearby sequences.

[0032] “Expression of a gene” or “expression of a nucleic acid” meanstranscription of DNA into RNA (optionally including modification of theRNA, e.g., splicing), translation of RNA into a polypeptide (possiblyincluding subsequent modification of the polypeptide, e.g.,posttranslational modification), or both transcription and translation,as indicated by the context.

[0033] The term “vector” refers to the means by which a nucleic acid canbe propagated and/or transferred between organisms, cells, or cellularcomponents. Vectors include plasmids, viruses, bacteriophage,pro-viruses, phagemids, transposons, and artificial chromosomes, and thelike, that replicate autonomously or can integrate into a chromosome ofa host cell. A vector can also be a naked RNA polynucleotide, a nakedDNA polynucleotide, a polynucleotide composed of both DNA and RNA withinthe same strand, a poly-lysine-conjugated DNA or RNA, apeptide-conjugated DNA or RNA, a liposome-conjugated DNA, or the like,that are not autonomously replicating. Most commonly, the vectors of thepresent invention are plasmids.

[0034] An “expression vector” is a vector, such as a plasmid, which iscapable of promoting expression as well as replication of a nucleic acidincorporated therein. Typically, the nucleic acid to be expressed is“operably linked” to a promoter and/or enhancer, and is subject totranscription regulatory control by the promoter and/or enhancer.

[0035] The term “host cell” means a cell which contains a heterologousnucleic acid, such as a vector, and supports the replication and/orexpression of the nucleic acid. Host cells can be prokaryotic cells suchas E. coli, or eukaryotic cells such as yeast, insect, amphibian, avianor mammalian cells, including human cells. Exemplary host cells in thecontext of the invention include HEp-2 cells and Vero cells.

[0036] The term “introduced” when referring to a heterologous orisolated nucleic acid refers to the transfer of a nucleic acid into aeukaryotic or prokaryotic cell where the nucleic acid can beincorporated into the genome of the cell (e.g., chromosome, plasmid,plastid or mitochondrial DNA), converted into an autonomous replicon, ortransiently expressed (e.g., transfected mRNA). The term includes suchmethods as “infection,” “transfection,” “transformation” and“transduction.” In the context of the invention a variety of methods canbe employed to introduce nucleic acids into host cells, includingelectroporation, calcium phosphate precipitation, lipid mediatedtransfection (lipofection), etc.

[0037] An “open reading frame” or “ORF” is a possible translationalreading frame of DNA or RNA (e.g., of a gene), which is capable of beingtranslated into a polypeptide. That is, the reading frame is notinterrupted by stop codons. However, it should be noted that the termORF does not necessarily indicate that the polynucleotide is, in fact,translated into a polypeptide.

[0038] A “polypeptide” is a polymer comprising two or more amino acidresidues (e.g., a peptide or a protein). The polymer can optionallycomprise modifications such as glycosylation or the like. The amino acidresidues of the polypeptide can be natural or non-natural and can beunsubstituted, unmodified, substituted or modified.

[0039] The term “recombinant” indicates that the material (e.g., avirus, a nucleic acid, or a protein) has been artificially orsynthetically (non-naturally) altered by human intervention. Thealteration can be performed on the material within, or removed from, itsnatural environment or state. For example, a “recombinant nucleic acid”is one that is made by recombining nucleic acids, e.g., during cloning,DNA shuffling or other procedures, or by chemical or other mutagenesis;a “recombinant polypeptide” or “recombinant protein” is a polypeptide orprotein which is produced by expression of a recombinant nucleic acid;and a “recombinant virus”, e.g., a recombinant respiratory syncytialvirus, is produced by the expression of a recombinant nucleic acid.

[0040] The term “isolated” refers to a biological material, such as avirus, a nucleic acid or a protein, which is substantially free fromcomponents that normally accompany or interact with it in its naturallyoccurring environment. The isolated material optionally comprisesmaterial not found with the material in its natural environment, e.g., acell. For example, if the material is in its natural environment, suchas a cell, the material has been placed at a location in the cell (e.g.,genome or genetic element) not native to a material found in thatenvironment. For example, a naturally occurring nucleic acid (e.g., acoding sequence, a promoter, an enhancer, etc.) becomes isolated if itis introduced by non-naturally occurring means to a locus of the genome(e.g., a vector, such as a plasmid or virus vector, or amplicon) notnative to that nucleic acid. Such nucleic acids are also referred to as“heterologous” nucleic acids. An isolated virus, for example, is in anenvironment (e.g., a cell culture system, or purified from cell culture)other than the native environment of wild-type virus (e.g., thenasopharynx of an infected individual).

[0041] The term “chimeric” or “chimera,” when referring to a virus,indicates that the virus includes genetic and/or polypeptide componentsderived from more than one parental viral strain or source. Similarly,the term “chimeric” or “chimera,” when referring to a viral protein,indicates that the protein includes polypeptide components (i.e., aminoacid subsequences) derived from more than one parental viral strain orsource.

[0042] An RSV “having an attenuated phenotype” or an “attenuated” RSVexhibits a substantially lower degree of virulence as compared to awild-type virus (e.g., one circulating naturally among human hosts). Anattenuated RSV typically exhibits a slower growth rate and/or a reducedlevel of replication (e.g., a peak titer, e.g., in cell culture, in ananimal model of infection, or in a human vacinee's nasopharynx, that isat least about ten fold, preferably at least about one hundred fold,less than that of a wild-type RSV).

[0043] An “immunologically effective amount” of RSV is an amountsufficient to enhance an individual's (e.g., a human's) own immuneresponse against a subsequent exposure to RSV. Levels of inducedimmunity can be monitored, e.g., by measuring amounts of neutralizingsecretory and/or serum antibodies, e.g., by plaque neutralization,complement fixation, enzyme-linked immunosorbent, or microneutralizationassay.

[0044] A “protective immune response” against RSV refers to an immuneresponse exhibited by an individual (e.g., a human) that is protectiveagainst serious lower respiratory tract disease (e.g., pneumonia and/orbronchiolitis) when the individual is subsequently exposed to and/orinfected with wild-type RSV. In some instances, the wild-type (e.g.,naturally circulating) RSV can still cause infection, particularly inthe upper respiratory tract (e.g., rhinitis), but it can not cause aserious infection. Typically, the protective immune response results indetectable levels of host engendered serum and secretory antibodies thatare capable of neutralizing virus of the same strain and/or subgroup(and possibly also of a different, non-vaccine strain and/or subgroup)in vitro and in vivo.

[0045] As used herein, an “antibody” is a protein comprising one or morepolypeptides substantially or partially encoded by immunoglobulin genesor fragments of immunoglobulin genes. The recognized immunoglobulingenes include the kappa, lambda, alpha, gamma, delta, epsilon and muconstant region genes, as well as myriad immunoglobulin variable regiongenes. Light chains are classified as either kappa or lambda. Heavychains are classified as gamma, mu, alpha, delta, or epsilon, which inturn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE,respectively. A typical immunoglobulin (antibody) structural unitcomprises a tetramer. Each tetramer is composed of two identical pairsof polypeptide chains, each pair having one “light” (about 25 kD) andone “heavy” chain (about 50-70 kD). The N-terminus of each chain definesa variable region of about 100 to 110 or more amino acids primarilyresponsible for antigen recognition. The terms variable light chain (VL)and variable heavy chain (VH) refer to these light and heavy chainsrespectively. Antibodies exist as intact immunoglobulins or as a numberof well-characterized fragments produced by digestion with variouspeptidases. Thus, for example, pepsin digests an antibody below thedisulfide linkages in the hinge region to produce F(ab)′2, a dimer ofFab which itself is a light chain joined to VH-CH1 by a disulfide bond.The F(ab)′2 may be reduced under mild conditions to break the disulfidelinkage in the hinge region thereby converting the (Fab′)2 dimer into aFab′ monomer. The Fab′ monomer is essentially a Fab with part of thehinge region (see, Fundamental Immunology, W. E. Paul, ed., Raven Press,N.Y. (1999), for a more detailed description of other antibodyfragments). While various antibody fragments are defined in terms of thedigestion of an intact antibody, one of skill will appreciate that suchFab′ fragments may be synthesized de novo either chemically or byutilizing recombinant DNA methodology. Thus, the term antibody, as usedherein, includes antibodies or fragments either produced by themodification of whole antibodies or synthesized de novo usingrecombinant DNA methodologies. Antibodies include, e.g., polyclonalantibodies, monoclonal antibodies, multiple or single chain antibodies,including single chain Fv (sFv or scFv) antibodies in which a variableheavy and a variable light chain are joined together (directly orthrough a peptide linker) to form a continuous polypeptide, andhumanized or chimeric antibodies.

[0046] An “antigenome” is a polynucleotide that is complementary(typically, perfectly complementary) to a single-stranded viral (e.g.,RSV) genome. Since RSV is a negative-sense RNA virus, the genome is the“antisense” strand, and the antigenome is the “sense” strand thatcorresponds to mRNA.

[0047] A variety of additional terms are defined or otherwisecharacterized herein.

DETAILED DESCRIPTION

[0048] The present invention provides the complete polynucleotidesequence of human RSV subgroup B strain 9320. The sequence of a B9320antigenomic cDNA is listed as SEQ ID NO:1. As will be evident, the RSVgenome is an RNA with a polynucleotide sequence complementary to that ofSEQ ID NO:1.

[0049] The B9320 genome comprises 10 transcriptional units encoding 11proteins. Amino acid sequences of the proteins are also provided: NS1 islisted as SEQ ID NO:2, NS2 as SEQ ID NO:3, N as SEQ ID NO:4, P as SEQ IDNO:5, M as SEQ ID NO:6, SH as SEQ ID NO:7, G as SEQ ID NO:12, F as SEQID NO:8, M2-1 as SEQ ID NO:9, M2-2 as SEQ ID NO:10, and L as SEQ IDNO:11.

[0050] The invention provides isolated or recombinant polynucleotidesand polypeptides comprising the novel B9320 sequences. Recombinant RSVcomprising the nucleic acids and/or polypeptides (e.g., attenuatedrecombinant RSV suitable for use in attenuated live viral vaccines) arealso provided.

[0051] Polynucleotides of the Invention

[0052] One aspect of the present invention provides isolated orrecombinant nucleic acids comprising a polynucleotide sequence of theinvention. Polynucleotide sequences of the invention include thepolynucleotide sequence represented by SEQ ID NO:1, with the caveat thatSEQ ID NOs:14-19, representing limited subsequences of RSV B9320, havebeen previously described (e.g., in GenBank accession numbers M73544 andS75820; Jin et al. (1998) Virology 251:206-214; and Cheng et al. (2001)Virology 283:59-68). Thus, for example, an isolated or recombinantnucleic acid comprising the polynucleotide sequence of SEQ ID NO:1 or acomplementary polynucleotide sequence thereof is a favored embodiment ofthe invention. An isolated or recombinant nucleic acid comprising atleast one unique polynucleotide subsequence of SEQ ID NO:1 (e.g., aunique coding subsequence) or a complementary polynucleotide sequencethereof, with the proviso that the unique polynucleotide subsequenceincludes at least one subsequence not included in SEQ ID NOs:14-19 or acomplementary polynucleotide sequence thereof, is another favoredembodiment. The unique polynucleotide subsequence can, for example,comprise at least 10 contiguous nucleotides of SEQ ID NO:1 or acomplementary polynucleotide sequence thereof (e.g., at least 20contiguous nucleotides, at least 50 contiguous nucleotides, at least 100contiguous nucleotides, at least 500 contiguous nucleotides, or even atleast 1000 contiguous nucleotides).

[0053] In addition to the sequences explicitly provided in theaccompanying sequence listing, polynucleotide sequences that are highlyrelated structurally and/or functionally are polynucleotides of theinvention. Thus, polynucleotide sequences of the invention include apolynucleotide sequence that hybridizes under stringent conditions oversubstantially the entire length of the polynucleotide sequence of SEQ IDNO:1 (or a complementary sequence thereof) with at least 2× a signal tonoise ratio (e.g., at least 5× or at least 10× the signal to noiseratio) that the polynucleotide sequence hybridizes to the polynucleotidesequence of SEQ ID NO:13 or a complementary polynucleotide sequencethereof. Polynucleotide sequences of the invention also include apolynucleotide sequence that hybridizes under stringent conditions oversubstantially the entire length of a polynucleotide subsequencecomprising at least 100 contiguous nucleotides of SEQ ID NO:1 or itscomplementary sequence (e.g., a unique subsequence) with at least 2× asignal to noise ratio (e.g., at least 5× or at least 10× the signal tonoise ratio) that the polynucleotide sequence hybridizes to thecorresponding subsequence of SEQ ID NO:13 or a complementarypolynucleotide sequence thereof (or, optionally, the correspondingsubsequence of a genome of another naturally occurring respiratorysyncytial virus or a complementary polynucleotide sequence thereof).

[0054] Similarly, polynucleotide sequences of the invention include apolynucleotide sequence encoding an amino acid sequence or uniquesubsequence selected from the group consisting of SEQ ID NOs:2-11 or anartificial conservative variation thereof.

[0055] In addition to the polynucleotide sequences of the invention,e.g., listed in SEQ ID NO:1, polynucleotide sequences that aresubstantially identical to a polynucleotide of the invention can be usedin the compositions and methods of the invention. Substantiallyidentical or substantially similar polynucleotide sequences are definedas polynucleotide sequences that are identical, on a nucleotide bynucleotide basis, with at least a subsequence of a referencepolynucleotide, e.g., selected from SEQ ID NO:1. Such polynucleotidescan include, e.g., insertions, deletions, and substitutions relative toSEQ ID NO:1. For example, isolated or recombinant nucleic acidscomprising polynucleotide sequences (or subsequences) having greaterthan 97.8% sequence identity to SEQ ID NO:1 or a complementarypolynucleotide sequence thereof, as determined by BLASTN using defaultparameters, with the proviso that the polynucleotide sequence includesat least one subsequence not selected from SEQ ID NOs:14-19, are favoredembodiments of the invention. For example, the polynucleotide sequences(or subsequences) can be at least 98.5% (e.g., at least 99.0%, at least99.5%, or more) identical to SEQ ID NO:1 or a complementarypolynucleotide sequence thereof.

[0056] The nucleic acids of the invention can be, e.g., single-strandedor double-stranded, and can be, e.g., a DNA (e.g., a cDNA), an RNA, oran artificial nucleic acid (e.g., a peptide nucleic acid). SEQ ID NO:1presents the DNA sequence of the antigenomic B9320 cDNA; however, itwill be understood that the complementary genomic polynucleotidesequence can readily be determined from SEQ ID NO:1 and that U in an RNAsequence corresponds to T in a DNA sequence.

[0057] Nucleic acids of the invention include nucleic acids encodingpolypeptides of the invention. In one general class of embodiments, thenucleic acid comprises at least one unique polynucleotide subsequence ofSEQ ID NO:1 (or a complementary polynucleotide sequence thereof)encoding at least 20 contiguous amino acid residues of any one of SEQ IDNOs:2-12 (e.g., at least 50, at least 100, at least 200, or morecontiguous amino acid residues). In one class of embodiments, the uniquepolynucleotide subsequence comprises at least one complete ORF,preferably at least one complete ORF encoding a polypeptide selectedfrom among SEQ ID NOs: 2-12. In some embodiments, the nucleic acidcomprises a plurality of complete open reading frames.

[0058] A nucleic acid of the invention optionally comprises at least oneartificially mutated nucleotide, e.g., at least one artificiallydeleted, inserted, and/or substituted nucleotide (e.g., in a noncodingregion, e.g., a C to G change at the fourth position of the antigenomicsequence, and/or in a coding region). For example, the nucleic acid cancomprise a plurality of artificially mutated nucleotides. Theartificially mutated nucleotide(s) can be introduced.by site-directedmutagenesis, chemical mutagenesis, or the like.

[0059] In certain embodiments, mutation of the polynucleotide sequenceresults in alteration of an encoded amino acid sequence. Thus, in oneclass of embodiments, at least one polypeptide encoded by the nucleicacid comprises at least one deleted, inserted, and/or substituted aminoacid residue (e.g., at least one conservatively or non-conservativelysubstituted amino acid residue). For example, the mutated nucleotide canbe located in an ORF encoding a polypeptide selected from SEQ IDNOs:2-12. Thus, in one class of example embodiments, the at least oneartificially mutated nucleotide is located in the open reading frameencoding the polypeptide of SEQ ID NO:12. The artificially mutatednucleotide(s) can comprise, e.g., a deletion, e.g., a deletion resultingin a deletion of one or more amino acid residues from the G proteinencoded by SEQ ID NO:12 (e.g., a deletion of residues 164-197), or adeletion resulting in a deletion of the open reading frame encoding G.In another class of example embodiments, the at least one artificiallymutated nucleotide is located in the open reading frame encoding thepolypeptide of SEQ ID NO:10. The artificially mutated nucleotide(s) cancomprise, e.g., a deletion, e.g., a deletion resulting in a deletion ofone or more amino acid residues from the M2-2 protein encoded by SEQ IDNO:10, or a deletion resulting in a deletion of the open reading frameencoding M2-2. As another example, at least one of the nucleotidesencoding amino acid residue 1, amino acid residue 4 and/or amino acidresidue 10 of M2-2 can be mutated (e.g., substituted or deleted, e.g.,forcing use of the second and/or third start codon and resulting in adeletion of amino acid residues 1-3 or 1-9 of M2-2).

[0060] The nucleic acids of the invention include chimeric nucleicacids, for example, a nucleic acid comprising at least one subsequenceof SEQ ID NO:1 or a complementary polynucleotide sequence thereof and atleast one polynucleotide subsequence from a different strain of virus.The subsequence of SEQ ID NO:1 is preferably a unique polynucleotidesubsequence that comprises at least 10 contiguous nucleotides of SEQ IDNO:1 or its complement and that includes at least one subsequence notincluded in SEQ ID NOs:14-19 or a complementary polynucleotide sequencethereof. The different strain of virus can be, e.g., a different strainof human RSV (e.g., A2, B1, or the like) or a different species of virus(e.g., another paramyxovirus, e.g., pneumonia virus of mice, bovine RSV,or metapneumovirus). Such chimeric nucleic acids can, for example,encode chimeric proteins and/or chimeric viruses (e.g., for use invaccines to induce a protective immune response against one or morestrains of RSV and/or another virus). For example, in certainembodiments, the nucleic acid comprises at least one complete openreading frame of SEQ ID NO:1 and at least one complete open readingframe of the different strain of virus.

[0061] Another class of embodiments provides vectors comprising thenucleic acids of the invention. Yet another class of embodimentsprovides a host cell into which such a vector has been introduced.

[0062] Polypeptides of the Invention

[0063] One aspect of the present invention provides RSV B9320polypeptides and variants thereof, for example, a polypeptide comprisingan amino acid sequence or subsequence that is encoded by a nucleic acidof the invention, with the proviso that the amino acid sequence orsubsequence is not encoded by SEQ ID NO:14.

[0064] One general class of embodiments provides isolated or recombinantpolypeptides comprising an amino acid sequence of the invention. Thus,for example, an isolated or recombinant polypeptide comprising an aminoacid sequence selected from the group consisting of SEQ ID NOs:2-11 is afavored embodiment of the invention. An isolated or recombinantpolypeptide comprising a unique amino acid subsequence comprising atleast 7 (e.g., at least 8, at least 10, at least 20, at least 50, ormore) contiguous amino acid residues of any one of SEQ ID NOs:2-11 isanother favored embodiment. Artificial conservative variations of aminoacid sequences or subsequences of the invention are also amino acidsequences of the invention. Such polypeptides are optionallyimmunogenic.

[0065] In addition to the amino acid sequences of the invention, e.g.,listed in SEQ ID NOs:2-11, amino acid sequences that are substantiallyidentical to an amino acid sequence of the invention can be used in thecompositions and methods of the invention. Substantially identical orsubstantially similar polypeptide sequences are defined as amino acidsequences that are identical, on an amino acid by amino acid basis, withat least a subsequence of a reference polypeptide, e.g., selected fromamong SEQ ID NOs:2-11. Such amino acid sequences can include, e.g.,insertions, deletions, and substitutions relative to SEQ ID NOs:2-11.For example, an isolated or recombinant polypeptide comprising an aminoacid sequence that is greater than 99.3% identical to SEQ ID NO:2, anamino acid sequence that is greater than 98.4% identical to SEQ ID NO:3,an amino acid sequence that is greater than 99.7% identical to SEQ IDNO:4, an amino acid sequence that is greater than 98.3% identical to SEQID NO:5, an amino acid sequence that is greater than 99.6% identical toSEQ ID NO:6, an amino acid sequence that is greater than 97.0% identicalto SEQ ID NO:7, an amino acid sequence that is greater than 99.3%identical to SEQ ID NO:8, an amino acid sequence that is greater than99.5% identical to SEQ ID NO:9, an amino acid sequence that is greaterthan 96.4% identical to SEQ ID NO:10, or an amino acid sequence that isgreater than 99.2% identical to SEQ ID NO:11, as determined by BLASTPusing default parameters, is a favored embodiment of the invention. Forexample, the isolated or recombinant polypeptide can comprise an aminoacid sequence (or subsequence) that is at least 99.5% identical to SEQID NO:2, at least 98.6% identical to SEQ ID NO:3, at least 99.9%identical to SEQ ID NO:4, at least 98.5% identical to SEQ ID NO:5, atleast 99.8% identical to SEQ ID NO:6, at least 97.2% identical to SEQ IDNO:7, at least 99.5% identical to SEQ ID NO:8, at least 99.7% identicalto SEQ ID NO:9, at least 96.6% identical to SEQ ID NO:10, or at least99.4% identical to SEQ ID NO:11, as determined by BLASTP using defaultparameters.

[0066] A polypeptide of the invention optionally comprises at least oneartificially altered amino acid, e.g., at least one deleted, inserted,and/or substituted amino acid. For example, the polypeptide can comprisea plurality of artificially altered amino acids.

[0067] One class of embodiments provides an isolated or recombinantpolypeptide comprising the amino acid sequence of SEQ ID NO:12 with adeletion of residues 164-197, or an artificial conservative variationthereof.

[0068] Methods of producing isolated or recombinant polypeptides formanother aspect of the invention. In the methods, a host cell into whicha vector (e.g., an expression vector, e.g., an expression vectorcomprising one or more ORFs, or a vector comprising an entire viralgenome or antigenome) comprising a nucleic acid of the invention hasbeen introduced is cultured in a suitable culture medium underconditions permitting expression of the nucleic acid. The polypeptide isthen isolated from the host cell and/or the medium. For example, thepolypeptide can be purified from the host cell and/or the medium suchthat the resulting purified polypeptide is enriched at least 5× ascompared to its initial state. Polypeptides produced according to themethods described herein are also features of the invention. Suchpolypeptides can comprise, e.g., subsequences (e.g., uniquesubsequences, immunogenic subsequences, etc.) of SEQ ID NOs:2-11 from afew amino acids (e.g., 7 or more, 10 or more, or 20 or more) up to thefull length proteins.

[0069] Determining Sequence Relationships

[0070] A variety of methods for determining relationships (e.g.,identity, similarity and/or homology) between two or more sequences,such as SEQ ID NO:1 and SEQ ID NO:13, are available and well known inthe art. The methods include manual alignment, computer assistedsequence alignment, and combinations thereof. A number of algorithms(which are generally computer implemented) for performing sequencealignment are widely available, or can be produced by one of skill.These methods include, e.g., the local homology algorithm of Smith andWaterman (1981) Adv. Appl. Math. 2:482; the homology alignment algorithmof Needleman and Wunsch (1970) J. Mol. Biol. 48:443; the search forsimilarity method of Pearson and Lipman (1988) Proc. Natl. Acad. Sci.(USA) 85:2444; and/or by computerized implementations of thesealgorithms (e.g., GAP, BESTFIT, FASTA, and TFASTA in the WisconsinGenetics Software Package Release 7.0, Genetics Computer Group, 575Science Dr., Madison, Wis.).

[0071] For example, software for performing sequence identity (andsequence similarity) analysis using the BLAST algorithm is described inAltschul et al. (1990) J. Mol. Biol. 215:403-410. This software ispublicly available, e.g., through the National Center for BiotechnologyInformation on the world wide web at ncbi.nlm.nih.gov. This algorithminvolves first identifying high scoring sequence pairs (HSPs) byidentifying short words of length W in the query sequence, which eithermatch or satisfy some positive-valued threshold score T when alignedwith a word of the same length in a database sequence. T is referred toas the neighborhood word score threshold. These initial neighborhoodword hits act as seeds for initiating searches to find longer HSPscontaining them. The word hits are then extended in both directionsalong each sequence for as far as the cumulative alignment score can beincreased. Cumulative scores are calculated using, for nucleotidesequences, the parameters M (reward score for a pair of matchingresidues; always >0) and N (penalty score for mismatching residues;always <0). For amino acid sequences, a scoring matrix is used tocalculate the cumulative score. Extension of the word hits in eachdirection are halted when: the cumulative alignment score falls off bythe quantity X from its maximum achieved value; the cumulative scoregoes to zero or below, due to the accumulation of one or morenegative-scoring residue alignments; or the end of either sequence isreached. The BLAST algorithm parameters W, T, and X determine thesensitivity and speed of the alignment. The BLASTN program (fornucleotide sequences) uses as defaults a wordlength (W) of 11, anexpectation (E) of 10, a cutoff of 100, M=5, N=−4, and a comparison ofboth strands. For amino acid sequences, the BLASTP (BLAST Protein)program uses as defaults a wordlength (W) of 3, an expectation (E) of10, and the BLOSUM62 scoring matrix (see, Henikoff and Henikoff (1989)Proc. Natl. Acad. Sci. USA 89:10915).

[0072] Additionally, the BLAST algorithm performs a statistical analysisof the similarity between two sequences (see, e.g., Karlin and Altschul(1993) Proc. Nat'l. Acad. Sci. USA 90:5873-5787). One measure ofsimilarity provided by the BLAST algorithm is the smallest sumprobability (P(N)), which provides an indication of the probability bywhich a match between two nucleotide or amino acid sequences would occurby chance. For example, a nucleic acid is considered similar to areference sequence (and, therefore, in this context, homologous) if thesmallest sum probability in a comparison of the test nucleic acid to thereference nucleic acid is less than about 0.1, or less than about 0.01,and or even less than about 0.001.

[0073] Another example of a useful sequence alignment algorithm isPILEUP. PILEUP creates a multiple sequence alignment from a group ofrelated sequences using progressive, pairwise alignments. It can alsoplot a tree showing the clustering relationships used to create thealignment. PILEUP uses a simplification of the progressive alignmentmethod of Feng and Doolittle (1987) J. Mol. Evol. 35:351-360. The methodused is similar to the method described by Higgins and Sharp (1989)CABIOS 5:151-153. The program can align, e.g., up to 300 sequences of amaximum length of 5,000 letters. The multiple alignment procedure beginswith the pairwise alignment of the two most similar sequences, producinga cluster of two aligned sequences. This cluster can then be aligned tothe next most related sequence or cluster of aligned sequences. Twoclusters of sequences can be aligned by a simple extension of thepairwise alignment of two individual sequences. The final alignment isachieved by a series of progressive, pairwise alignments. The programcan also be used to plot a dendogram or tree representation ofclustering relationships. The program is run by designating specificsequences and their amino acid or nucleotide coordinates for regions ofsequence comparison.

[0074] An additional example of an algorithm that is suitable formultiple DNA, or amino acid, sequence alignments is the CLUSTALW program(Thompson, J. D. et al. (1994) Nucl. Acids. Res. 22: 4673-4680).CLUSTALW performs multiple pairwise comparisons between groups ofsequences and assembles them into a multiple alignment based onhomology. Gap open and Gap extension penalties can be, e.g., 10 and 0.05respectively. For amino acid alignments, the BLOSUM algorithm can beused as a protein weight matrix. See, e.g., Henikoff and Henikoff (1992)Proc. Natl. Acad. Sci. USA 89: 10915-10919.

[0075] Nucleic Acid Hybridization

[0076] Similarity between nucleic acids can also be evaluated by“hybridization” between single stranded (or single stranded regions of)nucleic acids with complementary or partially complementarypolynucleotide sequences. Hybridization is a measure of the physicalassociation between nucleic acids, typically, in solution, or with oneof the nucleic acid strands immobilized on a solid support, e.g., amembrane, a bead, a chip, a filter, etc. Nucleic acid hybridizationoccurs based on a variety of well characterized physico-chemical forces,such as hydrogen bonding, solvent exclusion, base stacking and the like.Numerous protocols for nucleic acid hybridization are well known in theart. An extensive guide to the hybridization of nucleic acids is foundin Tijssen (1993) Laboratory Techniques in Biochemistry and MolecularBiologv—Hvbridization with Nucleic Acid Probes, part I, chapter 2,“Overview of principles of hybridization and the strategy of nucleicacid probe assays,” (Elsevier, N.Y.), as well as in Ausubel et al.Current Protocols in Molecular Biology (supplemented through 2003) JohnWiley & Sons, New York (“Ausubel”); Sambrook et al. Molecular Cloning—ALaboratory Manual (3rd Ed.), Vol. 1-3, Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y., 2001 (“Sambrook”), and Berger and Kimmel Guideto Molecular Cloning Techniques, Methods in Enzymology volume 152Academic Press, Inc., San Diego, Calif. (“Berger”). Hames and Higgins(1995) Gene Probes 1, IRL Press at Oxford University Press, Oxford,England (Hames and Higgins 1) and Hames and Higgins (1995) Gene Probes2, IRL Press at Oxford University Press, Oxford, England (Hames andHiggins 2) provide details on the synthesis, labeling, detection andquantification of DNA and RNA, including oligonucleotides.

[0077] Conditions suitable for obtaining hybridization, includingdifferential hybridization, are selected according to the theoreticalmelting temperature (T_(m)) between complementary and partiallycomplementary nucleic acids. Under a given set of conditions, e.g.,solvent composition, ionic strength, etc., the T_(m) is the temperatureat which the duplex between the hybridizing nucleic acid strands is 50%denatured. That is, the T_(m) corresponds to the temperaturecorresponding to the midpoint in transition from helix to random coil;it depends on length, nucleotide composition, and ionic strength forlong,stretches of nucleotides.

[0078] After hybridization, unhybridized nucleic acids can be removed bya series of washes, the stringency of which can be adjusted dependingupon the desired results. Low stringency washing conditions (e.g., usinghigher salt and lower temperature) increase sensitivity, but can producenonspecific hybridization signals and high background signals. Higherstringency conditions (e.g., using lower salt and higher temperaturethat is closer to the T_(m)) lower the background signal, typically withprimarily the specific signal remaining. See, also, Rapley, R. andWalker, J. M. eds., Molecular Biomethods Handbook (Humana Press, Inc.1998).

[0079] “Stringent hybridization wash conditions” or “stringentconditions” in the context of nucleic acid hybridization experiments,such as Southern and northern hybridizations, are sequence dependent,and are different under different environmental parameters. An extensiveguide to the hybridization of nucleic acids is found in Tijssen (1993),supra, and in Hames and Higgins 1 and Hames and Higgins 2, supra.

[0080] An example of stringent hybridization conditions forhybridization of complementary nucleic acids which have more than 100complementary residues on a filter in a Southern or northern blot is2×SSC, 50% formamide at 42° C., with the hybridization being carried outovernight (e.g., for approximately 20 hours). An example of stringentwash conditions is a 0.2×SSC wash at 65° C. for 15 minutes (seeSambrook, supra for a description of SSC buffer). Often, the washdetermining the stringency is preceded by a low stringency wash toremove signal due to residual unhybridized probe. An example lowstringency wash is 2×SSC at room temperature (e.g., 20° C. for 15minutes).

[0081] In general, a signal to noise ratio of at least 2× (or higher,e.g., at least 5×, 10×, 20×, 50×, 100×, or more) than that observed foran unrelated probe in the particular hybridization assay indicatesdetection of a specific hybridization. Detection of at least stringenthybridization between two sequences in the context of the presentinvention indicates relatively strong structural similarity to, e.g.,the nucleic acids of the present invention provided in the sequencelistings herein.

[0082] For purposes of the present invention, generally, “highlystringent” hybridization and wash conditions are selected to be about 5°C. or less lower than the thermal melting point (T_(m)) for the specificsequence at a defined ionic strength and pH (as noted below, highlystringent conditions can also be referred to in comparative terms).Target sequences that are closely related or identical to the nucleotidesequence of interest (e.g., “probe”) can be identified under stringentor highly stringent conditions. Lower stringency conditions areappropriate for sequences that are less complementary.

[0083] For example, in determining stringent or highly stringenthybridization (or even more stringent hybridization) and washconditions, the hybridization and wash conditions are graduallyincreased (e.g., by increasing temperature, decreasing saltconcentration, increasing detergent concentration and/or increasing theconcentration of organic solvents, such as formamide, in thehybridization or wash), until a selected set of criteria are met. Forexample, the hybridization and wash conditions are gradually increaseduntil a probe comprising one or more polynucleotide sequences of theinvention, e.g., sequences or unique subsequences selected from SEQ IDNO:1 and/or complementary polynucleotide sequences, binds to a perfectlymatched complementary target (again, a nucleic acid comprising one ormore nucleic acid sequences or subsequences selected from SEQ ID NO:1and/or complementary polynucleotide sequences thereof), with a signal tonoise ratio that is at least 2× (and optionally 5×, 10×, or 100× ormore) as high as that observed for hybridization of the probe to anunmatched target (e.g., a polynucleotide sequence comprising thecorresponding one or more sequences or subsequences selected from SEQ IDNO:13 and/or complementary polynucleotide sequences thereof), asdesired. Preferably, the sequences or subsequences are selected from aportion of SEQ ID NO:1 that includes at least a subsequence that is notincluded in SEQ ID NOs:14-19

[0084] Using the polynucleotides of the invention, or subsequencesthereof, novel target nucleic acids can be obtained; such target nucleicacids are also a feature of the invention. For example, such targetnucleic acids include sequences that hybridize under stringentconditions to a unique oligonucleotide probe corresponding to any of thepolynucleotides of the invention, e.g., SEQ ID NO:1.

[0085] Higher ratios of signal to noise can be achieved by increasingthe stringency of the hybridization conditions such that ratios of about15×, 20×, 30×, 50× or more are obtained. The particular signal willdepend on the label used in the relevant assay, e.g., a fluorescentlabel, a colorimetric label, a radioactive label, or the like.

[0086] Nucleic acids which do not hybridize to each other understringent conditions are still substantially identical if thepolypeptides which they encode are substantially identical. This occurs,e.g., when a copy of a nucleic acid is created using the maximum codondegeneracy permitted by the genetic code.

[0087] Defining Proteins by Immunoreactivity

[0088] Because the polypeptides of the invention provide a variety ofnew polypeptide sequences, the polypeptides also provide new structuralfeatures which can be recognized, e.g., in immunological assays. Thegeneration of antibodies or antisera which specifically bind thepolypeptides of the invention, as well as the polypeptides which arebound by such antibodies or antisera, and the antibodies or antiserathemselves, are a feature of the invention.

[0089] Thus, the proteins of the invention can also be identified byimmunoreactivity; e.g., a polypeptide of the invention can include anamino acid sequence or subsequence that is specifically bound by anantibody that specifically binds to an amino acid sequence orsubsequence encoded by SEQ ID NO:1, wherein the antibody does notspecifically bind to an amino acid sequence or subsequence encoded bySEQ ID NO:13 or SEQ ID NO:14 (or, optionally, to an amino acid sequenceor subsequence encoded by the genome of another naturally occurringrespiratory syncytial virus).

[0090] Methods of producing antibodies, performing immunoassays, and thelike are well known in the art. See e.g., the section entitled“Antibodies” below and references therein.

[0091] In one typical format, an immunoassay to identify a polypeptideof the invention uses a polyclonal antiserum which was raised againstone or more of the RSV 9320 polypeptides of the invention (e.g., apolypeptide comprising SEQ ID NOs:2-11 or SEQ ID NO:12 with a deletionof residues 164-197), or a substantial subsequence thereof (i.e., atleast about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 98% or more ofthe full length sequence provided). The full set of potentialpolypeptide immunogens derived from one or more of the RSV 9320polypeptides of the invention are collectively referred to below as “theimmunogenic polypeptides.” The resulting antisera is optionally selectedto have low cross-reactivity against the control RSV B1 polypeptidesand/or other known, e.g., naturally occurring, RSV polypeptides, and anysuch cross-reactivity is removed by immunoabsorption with one or more ofthe control RSV polypeptides, prior to use of the polyclonal antiserumin the immunoassay.

[0092] In order to produce antisera for use in an immunoassay, one ormore of the immunogenic polypeptides is produced and purified asdescribed herein. For example, recombinant protein can be produced in amammalian cell line. An inbred strain of mice (used in this assaybecause results are more reproducible due to the virtual geneticidentity of the mice) is immunized with the immunogenic polypeptide(s)in combination with a standard adjuvant, such as Freund's adjuvant, anda standard mouse immunization protocol (see Harlow and Lane (1988)Antibodies, A Laboratory Manual Cold Spring Harbor Press, New York, fora standard description of antibody generation, immunoassay formats andconditions that can be used to determine specific immunoreactivity).Alternatively, one or more synthetic or recombinant polypeptides derivedfrom the sequences disclosed herein is conjugated to a carrier proteinand used as an immunogen.

[0093] Polyclonal sera are collected and titered against the immunogenicpolypeptide(s) in an immunoassay, for example, a solid phase immunoassaywith one or more of the immunogenic polypeptides immobilized on a solidsupport. Polyclonal antisera with a titer of 10⁶ or greater areselected, pooled and subtracted with the control RSV polypeptides toproduce subtracted pooled titered polyclonal antisera.

[0094] The subtracted pooled titered polyclonal antisera are tested forcross reactivity against the control RSV polypeptides. Preferably atleast two of the immunogenic RSV 9320 polypeptides are used in thisdetermination, preferably in conjunction with at least two of thecontrol RSV polypeptides, to identify antibodies which are specificallybound by the immunogenic polypeptides(s).

[0095] In this comparative assay, discriminatory binding conditions aredetermined for the subtracted titered polyclonal antisera which resultin at least about a 5-10 fold higher signal to noise ratio for bindingof the titered polyclonal antisera to the immunogenic RSV 9320polypeptides as compared to binding to the control RSV polypeptides.That is, the stringency of the binding reaction is adjusted by theaddition of non-specific competitors, such as albumin or non-fat drymilk, or by adjusting salt conditions, temperature, or the like. Thesebinding conditions are used in subsequent assays for determining whethera test polypeptide is specifically bound by the pooled subtractedpolyclonal antisera. In particular, a test polypeptide which shows atleast a 2-5× higher signal to noise ratio than the control polypeptidesunder discriminatory binding conditions, and at least about a ½ signalto noise ratio as compared to the immunogenic polypeptide(s), sharessubstantial structural similarity or homology with the immunogenicpolypeptide(s) as compared to the control RSV polypeptides, and is,therefore, a polypeptide of the invention.

[0096] In another example, immunoassays in the competitive bindingformat are used for detection of a test polypeptide. For example, asnoted, cross-reacting antibodies are removed from the pooled antiseramixture by immunoabsorption with the control RSV polypeptides. Theimmunogenic polypeptide(s) are then immobilized to a solid support whichis exposed to the subtracted pooled antisera. Test proteins are added tothe assay to compete for binding to the pooled subtracted antisera. Theability of the test protein(s) to compete for binding to the pooledsubtracted antisera as compared to the immobilized protein(s) iscompared to the ability of the immunogenic polypeptide(s) added to theassay to compete for binding (the immunogenic polypeptides competeeffectively with the immobilized immunogenic polypeptides for binding tothe pooled antisera). The percent cross-reactivity for the test proteinsis calculated, using standard calculations.

[0097] In a parallel assay, the ability of the control proteins tocompete for binding to the pooled subtracted antisera is determined ascompared to the ability of the immunogenic polypeptide(s) to compete forbinding to the antisera. Again, the percent cross-reactivity for thecontrol polypeptides is calculated, using standard calculations. Wherethe percent cross-reactivity is at least 5-10× as high for the testpolypeptides, the test polypeptides are said to specifically bind thepooled subtracted antisera, and are, therefore, polypeptides of theinvention.

[0098] In general, the immunoabsorbed and pooled antisera can be used ina competitive binding immunoassay as described herein to compare anytest polypeptide to the immunogenic polypeptide(s). In order to makethis comparison, the two polypeptides are each assayed at a wide rangeof concentrations and the amount of each polypeptide required to inhibit50% of the binding of the subtracted antisera to the immobilized proteinis determined using standard techniques. If the amount of the testpolypeptide required is less than twice the amount of the immunogenicpolypeptide that is required, then the test polypeptide is said tospecifically bind to an antibody generated to the immunogenicpolypeptide, provided the amount is at least about 5-10× as high as fora control polypeptide.

[0099] As a final determination of specificity, the pooled antisera isoptionally fully immunosorbed with the immunogenic polypeptide(s)(rather than the control polypeptides) until little or no binding of theresulting immunogenic polypeptide subtracted pooled antisera to theimmunogenic polypeptide(s) used in the immunoabsorption is detectable.This fully immunosorbed antisera is then tested for reactivity with thetest polypeptide. If little or no reactivity is observed (i.e., no morethan 2× the signal to noise ratio observed for binding of the fullyimmunosorbed antisera to the immunogenic polypeptide), then the testpolypeptide is specifically bound by the antisera elicited by theimmunogenic protein.

[0100] Sequence Variations

[0101] Silent Variations

[0102] Due to the degeneracy of the genetic code, any of a variety ofnucleic acid sequences encoding polypeptides and/or viruses of theinvention are optionally produced, some which can bear lower levels ofsequence identity to the RSV nucleic acid and polypeptide sequences inthe figures. The following provides a typical codon table specifying thegenetic code, found in many biology and biochemistry texts. TABLE 1Codon Table Amino acids Codon Alanine Ala A GCA GCC GCG GCU Cysteine CysC UGC UGU Aspartic acid Asp D GAC GAU Glutamic acid Glu E GAA GAGPhenylalanine Phe F UUC UUU Glycine Gly G GGA GGC GGG GGU Histidine HisH CAC CAU Isoleucine Ile I AUA AUC AUU Lysine Lys K AAA AAG Leucine LeuL UUA UUG CUA CUC CUG CUU Methionine Met M AUG Asparagine Asn N AAC AAUProline Pro P CCA CCC CCG CCU Glutamine Gln Q CAA CAG Arginine Arg R AGAAGG CGA CGC CGG CGU Serine Ser S AGC AGU UCA UCC UCG UCU Threonine Thr TACA ACC ACG ACU Valine Val V GUA GUC GUG GUU Tryptophan Trp W UGGTyrosine Tyr Y UAC UAU

[0103] The codon table shows that many amino acids are encoded by morethan one codon. For example, the codons AGA, AGG, CGA, CGC, CGG, and CGUall encode the amino acid arginine. Thus, at every position in thenucleic acids of the invention where an arginine is specified by acodon, the codon can be altered to any of the corresponding codonsdescribed above without altering the encoded polypeptide. It isunderstood that U in an RNA sequence corresponds to T in a DNA sequence.

[0104] As an example, a nucleic acid sequence corresponding to the aminoacid sequence FEV (residues 164-166 of SEQ ID NO:12) is TTTGAAGTG. Asilent variation of this sequence includes TTCGAGGTA (also encodingFEV).

[0105] Such “silent variations” are one species of “conservativelymodified variations”, discussed below. One of skill will recognize thateach codon in a nucleic acid (except ATG, which is ordinarily the onlycodon for methionine, and TTG, which is ordinarily the only codon fortryptophan) can be modified by standard techniques to encode afunctionally identical polypeptide. Accordingly, each silent variationof a nucleic acid which encodes a polypeptide is implicit in anydescribed sequence. The invention, therefore, explicitly provides eachand every possible variation of a nucleic acid sequence encoding apolypeptide of the invention that could be made by selectingcombinations based on possible codon choices. These combinations aremade in accordance with the standard triplet genetic code (e.g., as setforth in Table 1, or as is commonly available in the art) as applied tothe nucleic acid sequence encoding an RSV polypeptide of the invention.All such variattions of every nucleic acid herein are specificallyprovided and described by consideration of the sequence in combinationwith the genetic code. One of skill is fully able to make these silentsubstitutions using the methods herein.

[0106] Conservative Variations

[0107] “Conservatively modified variations” or, simply, “conservativevariations” of a particular nucleic acid sequence or polypeptide arethose which encode identical or essntially indentical amino acidsequences. One of skill will recognize that individual substitutions,deletions or additions which alter, add or delete a single amino acid ora small percentage of amino acids (typically less than 5%, moretypically less than 4%, 2% or 1%) in an encoded sequence are“conservatively modified variations” where the alterations result in thedeletion of an amino acid, addition of an amino acid, or substitution ofan amino acid with a chemically similar amino acid.

[0108] Conservative substitution tables providing functionally similaramino acids are well known in the art. Table 2 sets forth six groupswhich contain amino acids that are “conservative substitutions” for oneanother. Alternative conservative substitution charts are available inthe art and can be used in a similar manner. TABLE 2 ConservativeSubstitution Groups 1 Alanine (A) Serine (S) Threonine (T) 2 Asparticacid (D) Glutamic acid (E) 3 Asparagine (N) Glutamine (Q) 4 Arginine (R)Lysine (K) 5 Isoleucine (I) Leucine (L) Methionine (M) Valine (V) 6Phenylalanine (F) Tyrosine (Y) Tryptophan (W)

[0109] Thus, “conservatively substituted variations” of a polypeptidesequence of the present invention include substitutions of a smallpercentage, typically less than 5%, more typically less than 2% or 1%,of the amino acids of the polypeptide sequence, with a conservativelyselected amino acid of the same conservative substitution group.

[0110] For example, a conservatively substituted variation of the RSVstrain B9320 M2-1 polypeptide in SEQ ID NO:9 will contain “conservativesubstitutions”, e.g., according to the six groups defined above, in upto about 10 residues (i.e., about 5% of the amino acids) in thefull-length polypeptide.

[0111] In a further example, if conservative substitutions werelocalized in the region corresponding to amino acids 10-12 of RSV 9320M2-1 (EIR), examples of conservatively substituted variations of thisregion include conservative substitutions of DLK or DMR (or any othersthat can be made according to Table 2) for EIR.

[0112] Listing of a protein sequence herein, in conjunction with theabove substitution table, provides an express listing of allconservatively substituted proteins.

[0113] Finally, the addition or deletion of sequences which do not alterthe encoded activity of a nucleic acid molecule, such as the addition ordeletion of a non-functional sequence, an epitope tag, a polyhistidinetag, GFP, or the like, is a conservative variation of the basic nucleicacid or polypeptide.

[0114] One of skill will appreciate that many conservative variations ofthe nucleic acid constructs which are disclosed yield a functionallyidentical construct. For example, as discussed above, owing to thedegeneracy of the genetic code, “silent substitutions” (i.e.,substitutions in a nucleic acid sequence which do not result in analteration in an encoded polypeptide) are an implied feature of everynucleic acid sequence which encodes an amino acid. Similarly,“conservative amino acid substitutions,” in which one or a few aminoacids in an amino acid sequence are substituted with different aminoacids with highly similar properties, are also readily identified asbeing highly similar to a disclosed construct. Such conservativevariations of each disclosed or claimed virus, nucleic acid or proteinare a feature of the present invention. Such conservative (e.g., silent)variations can be used, e.g., to produce antibodies for detection of orimmunoprotection against RSV.

[0115] Production of Viral Nucleic Acids

[0116] In the context of the invention, viral (e.g., RSV) nucleic acidsand/or proteins are manipulated according to well known molecularbiology techniques. Detailed protocols for numerous such procedures,including amplification, cloning, mutagenesis, transformation, and thelike, are described in, e.g., in Ausubel et al. Current Protocols inMolecular Biology (supplemented through 2003) John Wiley & Sons, NewYork (“Ausubel”); Sambrook et al. Molecular Cloning—A Laboratory Manual(3rd Ed.), Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y., 2001 (“Sambrook”), and Berger and Kimmel Guide to MolecularCloning Techniques, Methods in Enzymology volume 152 Academic Press,Inc., San Diego, Calif. (“Berger”).

[0117] In addition to the above references, protocols for in vitroamplification techniques, such as the polymerase chain reaction (PCR),the ligase chain reaction (LCR), Qβ-replicase amplification, and otherRNA polymerase mediated techniques (e.g., NASBA), useful e.g., foramplifying cDNA polynucleotides of the invention, are found in Mullis etal. (1987) U.S. Pat. No. 4,683,202; PCR Protocols A Guide to Methods andApplications (Innis et al. eds) Academic Press Inc. San Diego, Calif.(1990) (“Innis”); Arnheim and Levinson (1990) C&EN 36; The Journal OfNIH Research (1991) 3:81; Kwoh et al. (1989) Proc Natl Acad Sci USA 86,1173; Guatelli et al. (1990) Proc Natl Acad Sci USA 87:1874; Lomell etal. (1989) J Clin Chem 35:1826; Landegren et al. (1988) Science241:1077; Van Brunt (1990) Biotechnology 8:291; Wu and Wallace (1989)Gene 4: 560; Barringer et al. (1990) Gene 89:117, and Sooknanan andMalek (1995) Biotechnology 13:563. Additional methods, useful forcloning nucleic acids in the context of the present invention, includeWallace et al. U.S. Pat. No. 5,426,039. Improved methods of amplifyinglarge nucleic acids by PCR are summarized in Cheng et al. (1994) Nature369:684 and the references therein.

[0118] Certain polynucleotides of the invention, e.g., oligonucleotides,can be synthesized utilizing various solid-phase strategies includingmononucleotide- and/or trinucleotide-based phosphoramidite couplingchemistry. For example, nucleic acid sequences can be synthesized by thesequential addition of activated monomers and/or trimers to anelongating polynucleotide chain. See e.g., Caruthers, M. H. et al.(1992) Meth Enzymol 211:3.

[0119] In lieu of synthesizing the desired sequences, essentially anynucleic acid can be custom ordered from any of a variety of commercialsources, such as The Midland Certified Reagent Company (www.mcrc.com),The Great American Gene Company (www.genco.com), ExpressGen, Inc.(www.expressgen.com), QIAGEN (http://oligos.qiagen.com), and manyothers.

[0120] In addition, substitutions of selected amino acid residues inviral polypeptides can be accomplished by, e.g., site directedmutagenesis. For example, viral polypeptides with amino acidsubstitutions functionally correlated with desirable phenotypiccharacteristic, e.g., an attenuated phenotype, cold adaptation and/ortemperature sensitivity, can be produced by introducing specificmutations into a viral nucleic acid segment (e.g., a cDNA) encoding thepolypeptide. Methods for site directed mutagenesis are well known in theart, and are described, e.g., in Ausubel, Sambrook, and Berger, supra.Numerous kits for performing site directed mutagenesis are commerciallyavailable, e.g., the ExSite™ and Chameleon™ site directed mutagenesiskits (Stratagene, La Jolla), and can be used according to themanufacturer's instructions to introduce, e.g., one or more nucleotidesubstitutions specifying one or more amino acid substitutions into anRSV polynucleotide.

[0121] Various types of mutagenesis are optionally used in the presentinvention, e.g., to modify nucleic acids and encoded polypeptides and/orviruses to produce conservative or non-conservative variants. Anyavailable mutagenesis procedure can be used. Such mutagenesis proceduresoptionally include selection of mutant nucleic acids and polypeptidesfor one or more activity of interest. Procedures that can be usedinclude, but are not limited to: site-directed point mutagenesis, randompoint mutagenesis, in vitro or in vivo homologous recombination (DNAshuffling), mutagenesis using uracil containing templates,oligonucleotide-directed mutagenesis, phosphorothioate-modified DNAmutagenesis, mutagenesis using gapped duplex DNA, point mismatch repair,mutagenesis using repair-deficient host strains, restriction-selectionand restriction-purification, deletion mutagenesis, mutagenesis by totalgene synthesis, double-strand break repair, and many others known topersons of skill. In one embodiment, mutagenesis can be guided byinformation known about the naturally occurring molecule or altered ormutated naturally occurring molecules, e.g., sequence, sequencecomparisons, physical properties, crystal structure or the like. Inanother class of embodiments, modification is essentially random (e.g.,as in classical DNA shuffling).

[0122] Several of these procedures are set forth in Sambrook andAusubel, herein. Additional information on these procedures is found inthe following publications and the references cited therein: Arnold(1993) “Protein engineering for unusual environments” Current Opinion inBiotechnology 4:450-455; Bass et al. (1988) “Mutant Trp repressors withnew DNA-binding specificities” Science 242:240-245; Botstein and Shortle(1985) “Strategies and applications of in vitro mutagenesis” Science229:1193-1201; Carter et al. (1985) “Improved oligonucleotidesite-directed mutagenesis using M13 vectors” Nucl. Acids Res. 13:4431-4443; Carter (1986) “Site-directed mutagenesis” Biochem. J.237:1-7; Carter (1987) “Improved oligonucleotide-directed mutagenesisusing M13 vectors” Methods in Enzymol. 154: 382-403; Dale et al. (1996)“Oligonucleotide-directed random mutagenesis using the phosphorothioatemethod” Methods Mol. Biol. 57:369-374; Eghtedarzadeh and Henikoff (1986)“Use of oligonucleotides to generate large deletions” Nucl. Acids Res.14: 5115; Fritz et al. (1988) “Oligonucleotide-directed construction ofmutations: a gapped duplex DNA procedure without enzymatic reactions invitro” Nucl. Acids Res. 16: 6987-6999; Grundstrom et al. (1985)“Oligonucleotide-directed mutagenesis by microscale ‘shot-gun’ genesynthesis” Nucl. Acids Res. 13: 3305-3316; Kunkel (1987) “The efficiencyof oligonucleotide directed mutagenesis” in Nucleic Acids and MolecularBiology (Eckstein, F. and Lilley, D. M. J. eds., Springer Verlag,Berlin)); Kunkel (1985) “Rapid and efficient site-specific mutagenesiswithout phenotypic selection” Proc. Natl. Acad. Sci. USA 82:488-492;Kunkel et al. (1987) “Rapid and efficient site-specific mutagenesiswithout phenotypic selection” Methods in Enzymol. 154, 367-382; Krameret al. (1984) “The gapped duplex DNA approach tooligonucleotide-directed mutation construction” Nucl. Acids Res. 12:9441-9456; Kramer and Fritz (1987) “Oligonucleotide-directedconstruction of mutations via gapped duplex DNA” Methods in Enzymol.154:350-367; Kramer et al. (1984) “Point Mismatch Repair” Cell38:879-887; Kramer et al. (1988) “Improved enzymatic in vitro reactionsin the gapped duplex DNA approach to oligonucleotide-directedconstruction of mutations” Nucl. Acids Res. 16: 7207; Ling et al. (1997)“Approaches to DNA mutagenesis: an overview” Anal Biochem. 254(2):157-178; Lorimer and Pastan (1995) Nucleic Acids Res. 23, 3067-8;Mandecki (1986) “Oligonucleotide-directed double-strand break repair inplasmids of Escherichia coli: a method for site-specific mutagenesis”Proc. Natl. Acad. Sci. USA 83:7177-7181; Nakamaye and Eckstein (1986)“Inhibition of restriction endonuclease Nci I cleavage byphosphorothioate groups and its application to oligonucleotide-directedmutagenesis” Nucl. Acids Res. 14: 9679-9698; Nambiar et al. (1984)“Total synthesis and cloning of a gene coding for the ribonuclease Sprotein” Science 223: 1299-1301; Sakamar and Khorana (1988) “Totalsynthesis and expression of a gene for the a-subunit of bovine rod outersegment guanine nucleotide-binding protein (transducin)” Nucl. AcidsRes. 14: 6361-6372; Sayers et al. (1988) “Y-T Exonucleases inphosphorothioate-based oligonucleotide-directed mutagenesis” Nucl. AcidsRes. 16:791-802; Sayers et al. (1988) “Strand specific cleavage ofphosphorothioate-containing DNA by reaction with restrictionendonucleases in the presence of ethidium bromide” Nucl. Acids Res. 16:803-814; Sieber et al.(2001) Nature Biotechnology 19:456-460; Smith(1985) “In vitro mutagenesis” Ann. Rev. Genet. 19:423-462; Methods inEnzymol. 100: 468-500 (1983); Methods in Enzymol. 154: 329-350 (1987);Stemmer (1994) Nature 370, 389-91; Taylor et al. (1985) “The use ofphosphorothioate-modified DNA in restriction enzyme reactions to preparenicked DNA” Nucl. Acids Res. 13: 8749-8764; Taylor et al. (1985) “Therapid generation of oligonucleotide-directed mutations at high frequencyusing phosphorothioate-modified DNA” Nucl. Acids Res. 13: 8765-8787;Wells et al. (1986) “Importance of hydrogen-bond formation instabilizing the transition state of subtilisin” Phil. Trans. R. Soc.Lond. A 317: 415-423; Wells et al. (1985) “Cassette mutagenesis: anefficient method for generation of multiple mutations at defined sites”Gene 34:315-323; Zoller and Smith (1982) “Oligonucleotide-directedmutagenesis using M13-derived vectors: an efficient and generalprocedure for the production of point mutations in any DNA fragment”Nucleic Acids Res. 10:6487-6500; Zoller and Smith (1983)“Oligonucleotide-directed mutagenesis of DNA fragments cloned into M13vectors” Methods in Enzymol. 100:468-500; and Zoller and Smith (1987)“Oligonucleotide-directed mutagenesis: a simple method using twooligonucleotide primers and a single-stranded DNA template” Methods inEnzymol. 154:329-350. Additional details on many of the above methodscan be found in Methods in Enzymology Volume 154, which also describesuseful controls for trouble-shooting problems with various mutagenesismethods.

[0123] Vectors, Promoters and Expression Systems

[0124] The present invention includes recombinant constructsincorporating one or more of the nucleic acid sequences described above.Such constructs include a vector, for example, a plasmid, a cosmid, aphage, a virus, a bacterial artificial chromosome (BAC), a yeastartificial chromosome (YAC), etc., into which one or more of thepolynucleotide sequences of the invention, for example, SEQ ID NO:1 orsubsequences thereof, e.g., including at least one ORF selected from SEQID NO:1, has been inserted, in a forward or reverse orientation. Forexample, the inserted nucleic acid can include all or part of at leastone of the polynucleotide sequences of the invention. Typically thevector is selected based on the characteristics, e.g., size of theselected polynucleotide sequence, and on the intended use, e.g.,expression, amplification, etc. In a preferred embodiment, the constructfurther comprises regulatory sequences, including, for example, apromoter, operably linked to the sequence. Large numbers of suitablevectors and promoters are known to those of skill in the art and arecommercially available.

[0125] The polynucleotides of the present invention can be included inany one of a variety of vectors suitable for generating sense orantisense RNA, and optionally, polypeptide (or peptide) expressionproducts, e.g., selected from SEQ ID NOs:2-11. Such vectors includechromosomal, nonchromosomal and synthetic DNA sequences, e.g.,derivatives of SV40; bacterial plasmids; phage DNA; baculovirus; yeastplasmids; vectors derived from combinations of plasmids and phage DNA,viral DNA such as vaccinia, adenovirus, fowl pox virus, pseudorabies,adenovirus, adeno-associated virus, retroviruses, and many others, aswell as viral amplicon vectors. Any vector that is capable ofintroducing genetic material into a cell, and, if replication isdesired, which is replicable in the relevant host, can be used.

[0126] In an expression vector, the polynucleotide sequence, commonly asubsequence of SEQ ID NO:1, e.g., comprising an ORF, such as an ORFencoding a polypeptide (or peptide) selected from among SEQ ID NOs: 2-11(or variants thereof, e.g., conservative variations thereof), isphysically arranged in proximity and orientation to an appropriatetranscription control sequence (promoter, and optionally, one or moreenhancers) to direct mRNA synthesis. For example, a subsequence of SEQID NO:1, e.g., encoding a polypeptide selected from a subsequence of oneof SEQ ID NOs:2-11, can be inserted into an expression vector to produceantigenic peptide for the production of antibodies, e.g., for diagnosticor therapeutic purposes. That is, the polynucleotide sequence ofinterest is operably linked to an appropriate transcription controlsequence. Examples of such promoters include: LTR or SV40 promoter, E.coli lac or trp promoter, phage lambda P_(L) promoter, and otherpromoters known to control expression of genes in prokaryotic oreukaryotic cells or their viruses. The expression vector typically alsocontains a ribosome binding site for translation initiation, and atranscription terminator. The vector optionally includes appropriatesequences for amplifying expression. In addition, the expression vectorsoptionally comprise one or more selectable marker genes to provide aphenotypic trait for selection of transformed host cells, such asdihydrofolate reductase or neomycin resistance for eukaryotic cellculture, or such as tetracycline or ampicillin resistance in E. coli.

[0127] Where translation of polypeptide encoded by a nucleic acidcomprising a polynucleotide sequence of the invention is desired,additional translation specific initiation signals can improve theefficiency of translation. These signals can include, e.g., an ATGinitiation codon and adjacent sequences. In some cases, for example,full-length cDNA molecules or chromosomal segments including a codingsequence incorporating, e.g., a polynucleotide sequence of theinvention, a translation initiation codon and associated sequenceelements are inserted into the appropriate expression vectorsimultaneously with the polynucleotide sequence of interest. In suchcases, additional translational control signals are not required.However, in cases where only a polypeptide coding sequence, e.g.,encoding an amino acid sequence selected from among SEQ ID NOs:2-11 or aportion thereof, is inserted, exogenous translational control signals,including an ATG initiation codon is provided for expression of therelevant sequence. The initiation codon is put in the correct readingframe to ensure transcription of the polynucleotide sequence ofinterest. Exogenous transcriptional elements and initiation codons canbe of various origins, both natural and synthetic. The efficiency ofexpression can be enhanced by the inclusion of enhancers appropriate tothe cell system in use (Scharf D et al. (1994) Results Probl Cell Differ20:125-62; Bittner et al. (1987) Methods in Enzymol 153:516-544).

[0128] Polypeptide Production and Recovery

[0129] The present invention also relates to the introduction of vectorsincorporating the polynucleotides of the invention (e.g.,polynucleotides including all or part of one or more ORFs selected fromSEQ ID NO:1) into host cells and the production of polypeptides of theinvention, e.g., one or more polypeptide selected from SEQ ID NOs:2-11or subsequences thereof (e.g., unique subsequences thereof) byrecombinant techniques. Recombinant and/or isolated polypeptides encodedby the polynucleotides of the invention, e.g., SEQ ID NOs:2-11, orsubsequences (e.g., unique subsequences) thereof are used, for example,as antigens to produce antibodies in animal or human subjects. Forexample, antigenic polypeptides (or peptides) corresponding to all orpart of a polypeptide represented by SEQ ID NOs:2-11 can be injectedinto an experimental animal to produce antibodies specific for one ormore strains of RSV, as further described in the section entitled“Antibodies” below. Additionally, the antigenic polypeptides can beadministered, e.g., as a vaccine, to human subjects to elicit an immuneresponse specific for one or more strains of RSV. For example, such anelicited immune response can be a protective immune response.

[0130] To produce the polypeptides of the invention, host cells aregenetically engineered (e.g., transduced, transformed or transfected)with a vector, such as an expression vector, of this invention. Asdescribed above, the vector can be in the form of a plasmid, a viralparticle, a phage, etc. Examples of appropriate expression hostsinclude: bacterial cells, such as E. coli, Streptomyces, and Salmonellatyphimurium; fungal cells, such as Saccharomyces cerevisiae, Pichiapastoris, and Neurospora crassa; insect cells such as Drosophila andSpodoptera frugiperda; mammalian cells such as 3T3, COS, CHO, BHK, HEK293 or Bowes melanoma; plant cells, etc.

[0131] The engineered host cells can be cultured in conventionalnutrient media modified as appropriate for activating promoters,selecting transformants, or amplifying the inserted polynucleotidesequences. The culture conditions, such as temperature, pH and the like,are typically those previously used with the host cell selected forexpression, and will be apparent to those skilled in the art and in thereferences cited herein, including, e.g., Freshney (1994) Culture ofAnimal Cells, a Manual of Basic Technique, third edition, Wiley-Liss,New York and the references cited therein. In addition to Sambrook,Berger and Ausubel, details regarding cell culture can be found in Payneet al. (1992) Plant Cell and Tissue Culture in Liquid Systems John Wiley& Sons, Inc. New York, N.Y.; Gamborg and Phillips (eds) (1995) PlantCell, Tissue and Organ Culture; Fundamental Methods Springer Lab Manual,Springer-Verlag (Berlin Heidelberg N.Y.) and Atlas and Parks (eds) TheHandbook of Microbiological Media (1993) CRC Press, Boca Raton, Fla.

[0132] In bacterial systems, a number of expression vectors can beselected depending upon the use intended for the expressed product. Forexample, when large quantities of a polypeptide or fragments thereof areneeded for the production of antibodies, vectors which direct high levelexpression of fusion proteins that are readily purified are favorablyemployed. Such vectors include, but are not limited to, multifunctionalE. coli cloning and expression vectors such as BLUESCRIPT (Stratagene),in which the coding sequence of interest, e.g., a polynucleotide of theinvention as described above, can be ligated into the vector in-framewith sequences for the amino-terminal translation initiating Methionineand the subsequent 7 residues of beta-galactosidase producing acatalytically active beta galactosidase fusion protein; pIN vectors (VanHeeke and Schuster (1989) J Biol Chem 264:5503-5509); pET vectors(Novagen, Madison Wis.); and the like.

[0133] Similarly, in the yeast Saccharomyces cerevisiae a number ofvectors containing constitutive or inducible promoters such as alphafactor, alcohol oxidase and PGH can be used for production of thedesired expression products. For reviews, see Berger, Ausubel, and,e.g., Grant et al. (1987) Methods in Enzymology 153:516-544.

[0134] Vectors suitable for replication in mammalian cells are alsoknown in the art. Exemplary vectors include those derived from SV40,retroviruses, bovine papilloma virus, vaccinia virus, otherherpesviruses and adenovirus. Such suitable mammalian expression vectorsoptionally contain a promoter to mediate transcription of foreign DNAsequences and, optionally, an enhancer. Suitable promoters are known inthe art and include viral promoters such as those from SV40,cytomegalovirus (CMV), Rous sarcoma virus (RSV), adenovirus (ADV), andbovine papilloma virus (BPV).

[0135] The optional presence of an enhancer, combined with the promoterdescribed above, will typically increase expression levels. An enhanceris any regulatory DNA sequence that can stimulate transcription up to1000-fold when linked to endogenous or heterologous promoters, withsynthesis beginning at the normal mRNA start site. Enhancers are alsoactive when placed upstream or downstream from the transcriptioninitiation site, in either normal or flipped orientation, or at adistance of more than 1000 nucleotides from the promoter. See, e.g.,Maniatis (1987) Science 236:1237 and Alberts (1989) Molecular Biology ofthe Cell, 2nd Ed. (or later). Enhancers derived from viruses may beparticularly useful, because they typically have a broader host range.Examples include the SV40 early gene enhancer (see Dijkema (1985) EMBOJ. 4:761) and the enhancer/promoters derived from the long terminalrepeat (LTR) of the RSV (see Gorman (1982) Proc. Natl. Acad. Sci.79:6777) and from human cytomegalovirus (see Boshart (1985) Cell41:521). Additionally, some enhancers are regulatable and become activeonly in the presence of an inducer, such as a hormone or metal ion (seeSassone-Corsi and Borelli (1986) Trends Genet. 2:215); Maniatis (1987)Science 236:1237), In addition, the expression vector can and willtypically also include a termination sequence and poly(A) additionsequences which are operably linked to the heterologous coding sequence.

[0136] Sequences that cause amplification of the gene may also bedesirably included in the expression vector or in another vector that isco-translated with the expression vector, as are sequences which encodeselectable markers. Selectable markers for mammalian cells are known inthe art, and include, for example, thymidine kinase, dihydrofolatereductase (together with methotrexate as a DHFR amplifier),aminoglycoside phosphotransferase, hygromycin B phosphotransferase,asparagine synthetase, adenosine deaminase, metallothionien, andantibiotic resistant genes such as neomycin.

[0137] Mammalian cell lines available as hosts for expression are knownin the art and include many immortalized cell lines available from theAmerican Type Culture Collection (ATCC) as well as primary culturedcells and established cell lines, including but not limited to Vero,HEp-2, 3T3, COS, CHO, HeLa, BHK, MDCK, 293, WI38, Hep G2, MRC-5, andmany others.

[0138] Host cells containing the vectors (e.g., expression vectors)described above are also a feature of the invention. The host cell canbe a eukaryotic cell, such as a mammalian cell, a yeast cell, or a plantcell, or the host cell can be a prokaryotic cell, such as a bacterialcell. Introduction of the construct into the host cell can be effectedby calcium phosphate transfection, DEAE-Dextran mediated transfection,electroporation, encapsulation of the polynucleotide(s) in liposomes,direct microinjection of the DNA into nuclei, or other common techniques(see, e.g., Davis, L., Dibner, M., and Battey, I. (1986) Basic Methodsin Molecular Biology).

[0139] A host cell strain is optionally chosen for its ability tomodulate the expression of the inserted sequences or to process theexpressed protein in the desired fashion. Such modifications of theprotein include, but are not limited to, acetylation, carboxylation,glycosylation, phosphorylation, lipidation and acylation.Post-translational processing which cleaves a precursor form into amature form of the protein is sometimes important for correct insertion,folding and/or function. Different host cells have specific cellularmachinery and characteristic mechanisms for such post-translationalactivities and can be chosen to ensure the correct modification andprocessing of the introduced, foreign protein.

[0140] Host cells transformed with a nucleotide sequence encoding apolypeptide of the invention are optionally cultured under conditionssuitable for the expression and recovery of the encoded protein fromcell culture. The protein or fragment thereof produced by a recombinantcell can be secreted, membrane-bound, or contained intracellularly,depending on the sequence -and/or the vector used.

[0141] Following transduction of a suitable host cell line or strain andgrowth of the host cells to an appropriate cell density, the selectedpromoter is induced if necessary by appropriate means (e.g., temperatureshift or chemical induction) and cells are cultured for an additionalperiod. The secreted polypeptide product is then recovered from theculture medium. Alternatively, cells can be harvested by centrifugation,disrupted by physical or chemical means, and the resulting crude extractretained for further purification. Eukaryotic or microbial cellsemployed in expression of proteins can be disrupted by any convenientmethod, including freeze-thaw cycling, sonication, mechanicaldisruption, or use of cell lysing agents, or other methods, which arewell know to those skilled in the art.

[0142] Expressed polypeptides can be recovered and purified fromrecombinant cell cultures by any of a number of methods well known inthe art, including ammonium sulfate or ethanol precipitation, acidextraction, anion or cation exchange chromatography, phosphocellulosechromatography, hydrophobic interaction chromatography, affinitychromatography (e.g., using any of the tagging systems noted herein),hydroxylapatite chromatography, and lectin chromatography. Proteinrefolding steps can be used, as desired, in completing configuration ofthe mature protein. Finally, high performance liquid chromatography(HPLC) can be employed in the final purification steps. In addition tothe references noted above, a variety of purification methods are wellknown in the art, including, e.g., those set forth in Deutscher, Methodsin Enzymology Vol. 182: Guide to Protein Purification, Academic Press,Inc. N.Y. (1990); Sandana (1997) Bioseparation of Proteins, AcademicPress, Inc.; Bollag et al. (1996) Protein Methods, 2^(nd) EditionWiley-Liss, NY; Walker (1996) The Protein Protocols Handbook HumanaPress, NJ; Harris and Angal (1990) Protein Purification Applications: APractical Approach IRL Press at Oxford, Oxford, U.K.; Scopes (1993)Protein Purification: Principles and Practice 3^(rd) Edition SpringerVerlag, NY; Janson and Ryden (1998) Protein Purification: Principles,High Resolution Methods and Applications, Second Edition Wiley-VCH, NY;and Walker (1998) Protein Protocols on CD-ROM Humana Press, NJ.

[0143] Alternatively, cell-free transcription/translation systems can beemployed to produce polypeptides comprising an amino acid sequence orsubsequence encoded by the polynucleotides of the invention. A number ofsuitable in vitro transcription and translation systems are commerciallyavailable. A general guide to in vitro transcription and translationprotocols is found in Tymms (1995) In vitro Transcription andTranslation Protocols: Methods in Molecular Biology Volume 37, GarlandPublishing, NY. Cell free transcription/translation systems can beparticularly useful for the production of polypeptides, includingproteins for administration to human subjects.

[0144] In addition, the polypeptides, or subsequences thereof, e.g.,subsequences comprising antigenic peptides, can be produced manually orby using an automated system, by direct peptide synthesis usingsolid-phase techniques (see, e.g., Stewart et al. (1969) Solid-PhasePeptide Synthesis, W H Freeman Co, San Francisco; Merrifield J (1963) J.Am. Chem. Soc. 85:2149-2154). Exemplary automated systems include theApplied Biosystems 431A Peptide Synthesizer (Perkin Elmer, Foster City,Calif.). If desired, subsequences can be chemically synthesizedseparately, and combined using chemical methods to provide full-lengthpolypeptides.

[0145] Modified Amino Acids

[0146] Expressed polypeptides of the invention can contain one or moremodified amino acid. The presence of modified amino acids can beadvantageous in, for example, (a) increasing polypeptide serumhalf-life, (b) reducing polypeptide antigenicity or (c) increasingpolypeptide storage stability. Amino acid(s) are modified, for example,co-translationally or post-translationally during recombinant production(e.g., N-linked glycosylation at N-X-S/T motifs during expression inmammalian cells) or are modified by synthetic means (e.g., viaPEGylation).

[0147] Non-limiting examples of a modified amino acid include aglycosylated amino acid, a sulfated amino acid, a prenlyated (e.g.,famesylated, geranylgeranylated) amino acid, an acetylated amino acid,an acylated amino acid, a PEG-ylated amino acid, a biotinylated aminoacid, a carboxylated amino acid, a phosphorylated amino acid, and thelike, as well as amino acids modified by conjugation to, e.g., lipidmoieties or other organic derivatizing agents. References adequate toguide one of skill in the modification of amino acids are repletethroughout the literature. Example protocols are found in Walker (1998)Protein Protocols on CD-ROM Human Press, Towata, N.J.

[0148] Antibodies

[0149] The polypeptides of the invention can be used to produceantibodies specific for the polypeptides comprising amino acid sequencesor subsequences encoded by the polynucleotides of the invention.Antibodies specific for antigenic peptides encoded by, e.g., SEQ ID NO:1(e.g., SEQ ID NOs:2-11), and related variant polypeptides are useful,e.g., for diagnostic and therapeutic purposes, e.g., related to theactivity, distribution, and expression of target polypeptides.

[0150] Antibodies specific for the polypeptides of the invention can begenerated by methods well known in the art. Such antibodies can include,but are not limited to, polyclonal, monoclonal, chimeric, humanized,single chain, Fab fragments and fragments produced by an Fab expressionlibrary.

[0151] Polypeptides do not require biological activity for antibodyproduction. However, the polypeptide or oligopeptide is antigenic.Peptides used to induce specific antibodies typically have an amino acidsequence of at least about 5 amino acids, and often at least 10 or 20amino acids. Short stretches of a polypeptide, e.g., encoded by apolynucleotide of the invention such a sequence selected from SEQ IDNO:1 (such as a polypeptide selected from among SEQ ID NOs:2-11) canoptionally be fused with another protein, such as keyhole limpethemocyanin (KLH), and antibodies produced against the fusion protein orpolypeptide.

[0152] Numerous methods for producing polyclonal and monoclonalantibodies are known to those of skill in the art, and can be adapted toproduce antibodies specific for the polypeptides or peptides of theinvention. See, e.g., Coligan (1991) Current Protocols in ImmunologyWiley/Greene, NY; and Harlow and Lane (1989) Antibodies: A LaboratoryManual Cold Spring Harbor Press, NY; Stites et al. (eds.) Basic andClinical Immunology (4^(th) ed.) Lange Medical Publications, Los Altos,Calif., and references cited therein; Goding (1986) MonoclonalAntibodies: Principles and Practice (2d ed.) Academic Press, New York,N.Y.; Fundamental Immunology, e.g., 4^(th) Edition (or later),W. E. Paul(ed.), Raven Press, N.Y. (1998); and Kohler and Milstein (1975) Nature256: 495-497. Other suitable techniques for antibody preparation includeselection of libraries of recombinant antibodies in phage or similarvectors. See, Huse et al. (1989) Science 246: 1275-1281; and Ward, etal. (1989) Nature 341: 544-546. Specific monoclonal and polyclonalantibodies and antisera will usually bind with a K_(D) of at least about0.1 μM, preferably at least about 0.01 μM or better, and most typicallyand preferably, 0.001 μM or better.

[0153] For certain therapeutic applications (e.g., administration of anantibody or anitserum specific for one or more strains of RSV to providepassive immunity to a subject, e.g., a human, to prevent or decrease theseverity of RSV disease), humanized antibodies are desirable. Detailedmethods for preparation of humanized antibodies can be found in U.S.Pat. No. 5,482,856. Additional details on humanization and otherantibody production and engineering techniques can be found inBorrebaeck (ed) (1995) Antibody Engineering. 2^(nd) Edition Freeman andCompany, NY (Borrebaeck); McCafferty et al. (1996) Antibody Engineering,A Practical Approach IRL at Oxford Press, Oxford, England (McCafferty),and Paul (1995) Antibody Engineering Protocols Humana Press, Towata,N.J. (Paul). Additional details regarding specific procedures can befound, e.g., in Ostberg et al. (1983) Hybridoma 2: 361-367, Ostberg,U.S. Pat. No. 4,634,664, and Engelman et al. U.S. Pat. No. 4,634,666.

[0154] Diagnostic Assays

[0155] The novel nucleic acid sequences of the present invention can beused in diagnostic assays to detect RSV in a sample, to detect RSVB9320-like sequences, and to detect strain differences in clinicalisolates of RSV using either chemically synthesized or recombinant RSVB9320 polynucleotide fragments, e.g., selected from SEQ ID NO:1. Forexample, fragments of the novel B9320 sequences (SEQ ID NO:1) comprisingat least between 10 and 20 nucleotides can be used as primers to amplifynucleic acids using polymerase chain reaction (PCR) methods well knownin the art (e.g., reverse transcription-PCR) and as probes in nucleicacid hybridization assays to detect target genetic material such as RSVRNA in clinical specimens.

[0156] The novel RSV B9320 polynucleotide sequences can be used in theirentirety or as fragments to detect the presence of RNA sequences ortranscription products in cells, tissues, samples and the like usinghybridization techniques known in the art or in conjunction with one ofthe methods discussed herein. The probes can be either DNA or RNAmolecules, such as fragments of viral RNA, isolated restrictionfragments of cloned DNA, cDNAs, amplification products, transcripts, andoligonucleotides, and can vary in length from oligonucleotides as shortas about 10 nucleotides in length to viral RNA fragments or cDNAs inexcess of one or more kilobases. For example, in some embodiments, aprobe of the invention includes a polynucleotide sequence or subsequence(e.g., a unique subsequence) selected from SEQ ID NO:1 or sequencescomplementary thereto. Preferably the polynucleotide sequence (orsubsequence) is selected from a portion of SEQ ID NO:1 that includes atleast a subsequence that is not included in SEQ ID NOs:14-19.Alternatively, polynucleotide sequences that are variants of one of theabove designated sequences are used as probes. Most typically, suchvariants include one or a few nucleotide variations. For example, pairs(or sets) of oligonucleotides can be selected, in which the two (ormore) polynucleotide sequences are substantially identical variants ofeach other, wherein one polynucleotide sequence or set correspondsidentically to a first viral strain (e.g., B9320) and the othersequence(s) or set(s) correspond identically to additional viral strains(e.g., B1, A2, etc.). Such pairs of oligonucleotide probes areparticularly useful, for example, in the context of an allele specifichybridization experiment to determine the identity of an RSV virus orviral nucleic acid, e.g., for diagnostic or monitoring purposes. Inother applications, probes are selected that are more or less divergent,that is probes that are at least about 70% (or 80%, 90%, 95%, 98%, or99%) identical are selected.

[0157] The probes of the invention, e.g., as exemplified by uniquesubsequences selected from SEQ ID NO:1, can also be used to identifyadditional useful polynucleotide sequences (such as to characterizeadditional strains of RSV) according to procedures routine in the art.In one set of preferred embodiments, one or more probes, as describedabove, are utilized to screen libraries of expression products or clonedviral nucleic acids (i.e., expression libraries or genomic libraries) toidentify clones that include sequences identical to, or with significantsequence identity to SEQ ID NO:1. In turn, each of these identifiedsequences can be used to make probes, including pairs or sets of variantprobes as described above. It will be understood that in addition tosuch physical methods as library screening, computer assistedbioinformatic approaches, e.g., BLAST and other sequence homology searchalgorithms, and the like, can also be used for identifying relatedpolynucleotide sequences.

[0158] The probes of the invention are particularly useful for detectingthe presence and for determining the identity of RSV nucleic acids incells, tissues or other biological samples (e.g., a nasal wash orbronchial lavage). For example, the probes of the invention arefavorably utilized to determine whether a biological sample, such as asubject (e.g., a human subject) or model system (such as a cultured cellsample) has been exposed to, or become infected with, RSV. Detection ofhybridization of the selected probe to nucleic acids originating in(e.g., isolated from) the biological sample or model system isindicative of exposure to or infection with the virus (or a relatedvirus) from which the probe polynucleotide is selected. For example, apolynucleotide sequence that hybridizes preferentially to a subsequenceof SEQ ID NO:1 as compared to the corresponding subsequence of SEQ IDNO:13 (or the genome of another naturally occurring RSV strain) can beused to distinguish RSV B 9320 from RSV B1 (or another RSV strain).

[0159] It will be appreciated that probe design is influenced by theintended application. For example, where several allele-specificprobe-target interactions are to be detected in a single assay, e.g., ona single DNA chip, it is desirable to have similar melting temperaturesfor all of the probes. Accordingly, the length of the probes areadjusted so that the melting temperatures for all of the probes on thearray are closely similar (it will be appreciated that different lengthsfor different probes may be needed to achieve a particular T_(m) wheredifferent probes have different GC contents). Although meltingtemperature is a primary consideration in probe design, other factorsare optionally used to further adjust probe construction, such asselecting against primer self-complementarity and the like.

[0160] In other circumstances, e.g., relating to functional attributesof cells or organisms expressing the polynucleotides and polypeptides ofthe invention, probes that are polypeptides, peptides or antibodies arefavorably utilized. For example, polypeptides, polypeptide fragments andpeptides encoded by or having subsequences encoded by thepolynucleotides of the invention, e.g., SEQ ID NO:1, are favorably usedto identify and isolate antibodies or other binding proteins, e.g., fromphage display libraries, combinatorial libraries, polyclonal sera, andthe like.

[0161] Antibodies specific for a polypeptide subsequence encoded by anysubsequence (e.g., unique subsequence) or ORF of SEQ ID NO:1 arelikewise valuable as probes for evaluating expression products, e.g.,from cells or tissues. For example, suitable polypeptide sequences areselected from among the amino acid sequences represented by SEQ IDNOs:2-11. In addition, antibodies are particularly suitable forevaluating expression of proteins comprising amino acid subsequencesencoded by SEQ ID NO:1 (e.g., SEQ ID NOs:2-11), e.g., in a sample from asubject infected with or exposed to RSV. Antibodies can be directlylabeled with a detectable reagent as described below, or detectedindirectly by labeling of a secondary antibody specific for the heavychain constant region (i.e., isotype) of the specific antibody.Additional details regarding production of specific antibodies areprovided above in the section entitled “Antibodies.”

[0162] Labeling and Detecting Probes

[0163] Numerous methods are available for labeling and detection of thenucleic acid and polypeptide (or peptide or antibody) probes of theinvention. These include: 1) fluorescence (using, e.g., fluorescein,Cy-5, rhodamine or other fluorescent tags); 2) isotopic methods, e.g.,using end-labeling, nick translation, random priming, or PCR toincorporate radioactive isotopes into the probepolynucleotide/oligonucleotide; 3) chemifluorescence, e.g., usingalkaline phosphatase and the substrate AttoPhos (Amersham) or othersubstrates that produce fluorescent products; 4) chemiluminescence(e.g., using either horseradish peroxidase and/or alkaline phosphatasewith substrates that produce photons as breakdown products; kitsproviding reagents and protocols are available from such commercialsources as Amersham, Boehringer-Mannheim, and Life TechnologieslGibcoBRL); and, 5) colorimetric methods (again using, e.g., horseradishperoxidase and/or alkaline phosphatase with substrates that produce acolored precipitate; kits are available from Life Technologies/GibcoBRL, and Boehringer-Mannheim). Other methods for labeling and detectionwill be readily apparent to one skilled in the art.

[0164] More generally, a probe can be labeled with any compositiondetectable by spectroscopic, photochemical, biochemical, immunochemical,electrical, optical, chemical or other available means. Useful labels inthe present invention include spectral labels such as fluorescent dyes(e.g., fluorescein isothiocyanate, Texas red, rhodamine, and the like),radiolabels (e.g., ³H, ¹²⁵I, ³⁵S, ¹⁴C, ³²P, ³³P, etc.), enzymes (e.g.,horse-radish peroxidase, alkaline phosphatase, etc.), spectralcalorimetric labels such as colloidal gold or colored glass or plastic(e.g. polystyrene, polypropylene, latex, etc.) beads. The label may becoupled directly or indirectly to a component of the detection assay(e.g., a probe, such as an oligonucleotide, isolated DNA, amplicon,restriction fragment, or the like) according to methods well known inthe art. As indicated above, a wide variety of labels may be used, withthe choice of label depending on sensitivity required, ease ofconjugation with the compound, stability requirements, availableinstrumentation, and disposal provisions. In general, a detector whichmonitors a probe-target nucleic acid hybridization is adapted to theparticular label which is used. Typical detectors includespectrophotometers, phototubes and photodiodes, microscopes,scintillation counters, cameras, film and the like, as well ascombinations thereof. Examples of suitable detectors are widelyavailable from a variety of commercial sources known to persons ofskill. Commonly, an optical image of a substrate comprising a nucleicacid array with particular set of probes bound to the array is digitizedfor subsequent computer analysis.

[0165] Because incorporation of radiolabeled nucleotides into nucleicacids is straightforward, this detection represents one favorablelabeling strategy. Exemplary technologies for incorporating radiolabelsinclude end-labeling with a kinase or phosphatase enzyme, nicktranslation, incorporation of radio-active nucleotides with a polymeraseand many other well known strategies.

[0166] Fluorescent labels are desirable, having the advantage ofrequiring fewer precautions in handling, and being amenable tohigh-throughput visualization techniques. Preferred labels are typicallycharacterized by one or more of the following: high sensitivity, highstability, low background, low environmental sensitivity and highspecificity in labeling. Fluorescent moieties, which are incorporatedinto the labels of the invention, are generally are known, includingTexas red, fluorescein isothiocyanate, rhodamine, etc. Many fluorescenttags are commercially available from SIGMA chemical company (SaintLouis, Mo.), Molecular Probes (Eugene, Oreg.), R&D systems (Minneapolis,Minn.), Pharmacia LKB Biotechnology (Piscataway, N.J.), CLONTECHLaboratories, Inc. (Palo Alto, Calif.), Chem Genes Corp., AldrichChemical Company (Milwaukee, Wis.), Glen Research, Inc., GIBCO BRL LifeTechnologies, Inc. (Gaithersberg, Md.), Fluka Chemica-BiochemikaAnalytika (Fluka Chemie AG, Buchs, Switzerland), and Applied Biosystems(Foster City, Calif.) as well as other commercial sources known to oneof skill. Similarly, moieties such as digoxygenin and biotin, which arenot themselves fluorescent but are readily used in conjunction withsecondary reagents, i.e., anti-digoxygenin antibodies, avidin (orstreptavidin), that can be labeled, are suitable as labeling reagents inthe context of the probes of the invention.

[0167] The label is coupled directly or indirectly to a molecule to bedetected (a product, substrate, enzyme, or the like) according tomethods well known in the art. As indicated above, a wide variety oflabels are used, with the choice of label depending on the sensitivityrequired, ease of conjugation of the compound, stability requirements,available instrumentation, and disposal provisions. Non radioactivelabels are often attached by indirect means. Generally, a ligandmolecule (e.g., biotin) is covalently bound to a nucleic acid such as aprobe, primer, amplicon, or the like. The ligand then binds to ananti-ligand (e.g., streptavidin) molecule which is either inherentlydetectable or covalently bound to a signal system, such as a detectableenzyme, a fluorescent compound, or a chemiluminescent compound. A numberof ligands and anti-ligands can be used. Where a ligand has a naturalanti-ligand, for example, biotin, thyroxine, and cortisol, it can beused in conjunction with labeled anti-ligands. Alternatively, anyhaptenic or antigenic compound can be used in combination with anantibody. Labels can also be conjugated directly to signal generatingcompounds, e.g., by conjugation with an enzyme or fluorophore orchromophore. Enzymes of interest as labels will primarily be hydrolases,particularly phosphatases, esterases and glycosidases, oroxidoreductases, particularly peroxidases. Fluorescent compounds includefluorescein and its derivatives, rhodamine and its derivatives, dansyl,umbelliferone, etc. Chemiluminescent compounds include luciferin, and2,3-dihydrophthalazinediones, e.g., luminol. Means of detecting labelsare well known to those of skill in the art. Thus, for example, wherethe label is a radioactive label, means for detection include ascintillation counter or photographic film as in autoradiography. Wherethe label is optically detectable, typical detectors includemicroscopes, cameras, phototubes and photodiodes and many otherdetection systems which are widely available.

[0168] Production of Recombinant Virus

[0169] Negative strand RNA viruses can be genetically engineered andrecovered using a recombinant reverse genetics approach (U.S. Pat. No.5,166,057 to Palese et al.). Although this method was originally appliedto engineer influenza viral genomes (Luytjes et al. (1989) Cell59:1107-1113; Enami et al. (1990) Proc. Natl. Acad. Sci. USA92:11563-11567), it has been successfully applied to a wide variety ofsegmented and nonsegmented negative strand RNA viruses, e.g., rabies(Schnell et al. (1994) EMBO J. 13: 4195-4203); VSV (Lawson et al. (1995)Proc. Natl. Acad. Sci. USA 92: 4477-4481); measles virus (Radecke etal.(1995) EMBO J. 14:5773-5784); rinderpest virus (Baron and Barrett(1997) J.Virol. 71: 1265-1271); human parainfluenza virus (Hoffman andBanerjee (1997) J. Virol. 71: 3272-3277; Dubin et al. (1997) Virology235:323-332); SV5 (He et al. (1997) Virology 237:249-260); caninedistemper virus (Gassen et al. (2000) J. Virol. 74:10737-44); and Sendaivirus (Park et al. (1991) Proc. Natl. Acad. Sci. USA 88: 5537-5541; Katoet al. (1996) Genes to Cells 1:569-579).

[0170] Recently, a system for producing recombinant subgroup A RSV(e.g., attenuated recombinant RSV suitable for vaccine production) hasbeen described by the inventors and coworkers in WO 02/44334 by Jin etal. entitled “Recombinant RSV virus expression systems and vaccines,”the disclosure of which is incorporated herein in its entirety. Rescueof subgroup A RSV has also been described, e.g., in Jin et al. (1998)Virology 251:206-214 and Collins et al. (1995) Proc. Natl. Acad. Sci.USA 92:11563-11567. (See also e.g., Jin et al. (2000) J. Virol.74:74-82; Jin et al. (2000) Virology 273:210-218; Cheng et al. (2001)Virology 283:59-68; Tang et al. (2001) J. Virol. 75:11328-11335; U.S.patent application Ser. No. 60/444,287 (filed on Jan. 31, 2003) by Jinet al. entitled “Functional mutations in respiratory syncytial virus”;and U.S. patent application Ser. No. 10/672,302 (filed on Sep. 26, 2003)by Jin et al. entitled “Functional mutations in respiratory syncytialvirus.”) Rescue of subgroup B RSV is briefly described below and in theexamples herein.

[0171] In brief, recombinant RSV incorporating the nucleic acids of thisinvention are generated, for example, in a suitable cell line (e.g.,Vero or Hep-2 cells) by transfection of the cells with an antigenomiccDNA. Typically, the antigenomic cDNA is flanked by a T7 RNA polymerasepromoter and a hepatitis delta virus ribozyme plus the T7transcriptional terminator. Plasmids expressing the viral N, P, and Lproteins (and optionally also the M2-1 protein) are also introduced intothe cells, and a T7 RNA polymerase is typically expressed in thetransfected cells (e.g., by infection of the cells with a modifiedvaccinia virus Ankara expressing T7 RNA polymerase). Recombinant RSV canalso be produced by infection of suitable cells with previously isolatedrecombinant virus. Techniques for propagation, separation from host cellcellular components, and/or further purification of RSV are well knownto those skilled in the art.

[0172] Methods of producing recombinant RSV are a feature of theinvention. In the methods, a host cell into which a vector comprising anucleic acid of the invention has been introduced is cultured in asuitable culture medium under conditions permitting expression of thenucleic acid (e.g., coexpression of RSV N, P, and L and optionally M2-1and/or T7 RNA polymerase). The recombinant respiratory syncytial virusis then isolated from the host cell and/or the medium. Typically, thenucleic acid comprises an entire RSV genome or antigenome.Alternatively, the nucleic acid can comprise a portion of an RSV genomeor antigenome (e.g., one or more open reading frames encoding proteinsto be assembled with an RSV genome from another source to form therecombinant virus).

[0173] Recombinant RSV (e.g., attenuated recombinant RSV) producedaccording to the methods described herein are also a feature of theinvention, as are recombinant RSV (e.g., attenuated recombinant RSV)comprising one or more nucleic acids and/or polypeptides of theinvention.

[0174] Cell Culture

[0175] Typically, propagation of a recombinant virus (e.g., recombinantRSV) is accomplished in the media compositions in which the host cell iscommonly cultured. Suitable host cells for the replication of RSVinclude, e.g., Vero cells and HEp-2 cells. Typically, cells are culturedin a standard commercial culture medium, such as Dulbecco's modifiedEagle's medium supplemented with serum (e.g., 10% fetal bovine serum),or in serum free medium, under controlled humidity and CO₂ concentrationsuitable for maintaining neutral buffered pH (e.g., at pH between 7.0and 7.2). Optionally, the medium contains antibiotics to preventbacterial growth, e.g., penicillin, streptomycin, etc., and/oradditional nutrients, such as L-glutamine, sodium pyruvate,non-essential amino acids, additional supplements to promote favorablegrowth characteristics, e.g., trypsin, β-mercaptoethanol, and the like.

[0176] Procedures for maintaining mammalian cells in culture have beenextensively reported, and are known to those of skill in the art.General protocols are provided, e.g., in Freshney (1983) Culture ofAnimal Cells: Manual of Basic Technique, Alan R. Liss, New York; Paul(1975) Cell and Tissue Culture, 5^(th) ed., Livingston, Edinburgh; Adams(1980) Laboratory Techniques in Biochemistry and Molecular Biology-CellCulture for Biochemists, Work and Burdon (eds.) Elsevier, Amsterdam.Additionally, variations in such procedures adapted to the presentinvention are readily determined through routine experimentation.

[0177] Cells for production of RSV can be cultured in serum-containingor serum free medium. In some cases, e.g., for the preparation ofpurified viruses, it is desirable to grow the host cells in serum freeconditions. For example, cells can be grown to the desired density inserum-containing medium, infected, and then maintained in serum-freemedium. Cells can be cultured in small scale, e.g., less than 25 mlmedium, culture tubes or flasks or in large flasks with agitation, inrotator bottles, or on microcarrier beads (e.g., DEAE-Dextranmicrocarrier beads, such as Dormacell, Pfeifer & Langen; Superbead, FlowLaboratories; styrene copolymer-tri-methylamine beads, such as Hillex,SoloHill, Ann Arbor) in flasks, bottles or reactor cultures.Microcarrier beads are small spheres (in the range of 100-200 microns indiameter) that provide a large surface area for adherent cell growth pervolume of cell culture. For example a single liter of medium can includemore than 20 million microcarrier beads providing greater than 8000square centimeters of growth surface. For commercial production ofviruses, e.g., for vaccine production, it is often desirable to culturethe cells in a bioreactor or fermenter. Bioreactors are available involumes from under 1 liter to in excess of 100 liters, e.g., Cyto3Bioreactor (Osmonics, Minnetonka, Minn.); NBS bioreactors (New BrunswickScientific, Edison, N.J.); and laboratory and commercial scalebioreactors from B. Braun Biotech International (B. Braun Biotech,Melsungen, Germany).

[0178] Other useful references, e.g. for cell isolation and culture(e.g., of bacterial cells containing recombinant nucleic acids, e.g.,for subsequent nucleic acid isolation) include Freshney (1994) Cultureof Animal Cells, a Manual of Basic Technique, third edition, Wiley-Liss,New York and the references cited therein; Payne et al. (1992) PlantCell and Tissue Culture in Liquid Systems John Wiley & Sons, Inc. NewYork, N.Y.; Gamborg and Phillips (eds) (1995) Plant Cell, Tissue andOrgan Culture; Fundamental Methods Springer Lab Manual, Springer-Verlag(Berlin Heidelberg N.Y.) and Atlas and Parks (eds) The Handbook ofMicrobiological Media (1993) CRC Press, Boca Raton, Fla.

[0179] Introduction of Vectors Into Host Cells

[0180] Vectors, e.g., vectors incorporating RSV polynucleotides, areintroduced (e.g., transfected) into host cells according to methods wellknown in the art for introducing heterologous nucleic acids intoeukaryotic cells, including, e.g., calcium phosphate co-precipitation,electroporation, microinjection, lipofection, and transfection employingpolyamine transfection reagents. For example, vectors, e.g., plasmids,can be transfected into host cells, e.g., Vero cells or Hep-2 cells,using the transfection reagent LipofectACE or Lipofectamine 2000(Invitrogen) according to the manufacturer's instructions.Alternatively, electroporation can be employed to introduce vectorsincorporating RSV genome segments into host cells.

[0181] Model Systems

[0182] Attenuated RSV, e.g. those described herein comprising all orpart of SEQ ID NO:1 or variations thereof, can be tested in in vitro andin vivo models to confirm adequate attenuation, genetic stability,and/or immunogenicity for vaccine use. In in vitro assays, e.g.,replication in cultured cells, the virus can be tested, e.g., forgenetic stability, temperature sensitivity of virus replication and/or asmall plaque phenotype. RSV can be further tested in animal models ofinfection. A variety of animal models, e.g., primate (e.g., chimpanzee,African green monkey) and rodent (e.g., cotton rat), are known in theart, as described briefly herein and in U.S. Pat. No. 5,922,326 toMurphy et al. (Jul. 13, 1999) entitled “Attenuated respiratory syncytialvirus compositions”; U.S. Pat. No. 4,800,078; Meignier et al. eds.(1991) Animal Models of Respiratory Syncvtial Virus Infection, MerieuxFoundation Publication; Prince et al. (1985) Virus Res. 3:193-206;Richardson et al. (1978) J. Med. Virol. 3:91-100; Wright et al. Infect.Immun. (1982) 37:397-400; and Crowe et al. (1993) Vaccine 11:1395-1404.

[0183] Methods and Compositions for Prophylactic Administration ofVaccines

[0184] One aspect of the invention provides immunogenic compositions(e.g., vaccines) comprising an immunologically effective amount of arecombinant RSV of the invention (e.g., an attenuated live recombinantRSV), an immunologically effective amount of a polypeptide of theinvention, and/or an immunologically effective amount of a nucleic acidof the invention.

[0185] A related aspect of the invention provides methods forstimulating the immune system of an individual to produce a protectiveimmune response against respiratory syncytial virus. In the methods, animmunologically effective amount of a recombinant RSV of the invention,an immunologically effective amount of a polypeptide of the invention,and/or an immunologically effective amount of a nucleic acid of theinvention is administered to the individual in a physiologicallyacceptable carrier.

[0186] The RSV, polypeptides, and nucleic acids of the invention can beadministered prophylactically in an appropriate carrier or excipient tostimulate an immune response specific for one or more strains of RSV.Typically, the carrier or excipient is a pharmaceutically acceptablecarrier or excipient, such as sterile water, aqueous saline solution,aqueous buffered saline solutions, aqueous dextrose solutions, aqueousglycerol solutions, ethanol, or combinations thereof. The preparation ofsuch solutions insuring sterility, pH, isotonicity, and stability iseffected according to protocols established in the art. Generally, acarrier or excipient is selected to minimize allergic and otherundesirable effects, and to suit the particular route of administration,e.g., subcutaneous, intramuscular, intranasal, oral, topical, etc. Theresulting aqueous solutions can e.g., be packaged for use as is orlyophilized, the lyophilized preparation being combined with a sterilesolution prior to administration

[0187] Generally, the RSV (or RSV components) of the invention areadministered in a quantity sufficient to stimulate an immune responsespecific for one or more strains of RSV (e.g., an immunologicallyeffective amount of RSV or an RSV component is administered).Preferably, administration of RSV elicits a protective immune response.Dosages and methods for eliciting a protective anti-viral immuneresponse, adaptable to producing a protective immune response againstRSV, are known to those of skill in the art. See, e.g., U.S. Pat. No.5,922,326; Wright et al. (1982) Infect. Immun. 37:397-400; Kim et al.(1973) Pediatrics 52:56-63; and Wright et al. (1976) J. Pediatr.88:931-936. For example, virus can be provided in the range of about10³-10⁶ pfu (plaque forming units) per dose administered (e.g., 10⁴-10⁵pfu per dose administered). Typically, the dose will be adjusted basedon, e.g., age, physical condition, body weight, sex, diet, mode and timeof administration, and other clinical factors. The prophylactic vaccineformulation can be systemically administered, e.g., by subcutaneous orintramuscular injection using a needle and syringe or a needlelessinjection device. Preferably, the vaccine formulation is administeredintranasally, e.g., by drops, aerosol (e.g., large particle aerosol(greater than about 10 microns)), or spray into the upper respiratorytract. While any of the above routes of delivery results in a protectivesystemic immune response, intranasal administration confers the addedbenefit of eliciting mucosal immunity at the site of entry of the virus.For intranasal administration, attenuated live virus vaccines are oftenpreferred, e.g., an attenuated, cold adapted and/or temperaturesensitive recombinant RSV, e.g., a chimeric recombinant RSV. As analternative or in addition to attenuated live virus vaccines, killedvirus vaccines, nucleic acid vaccines, and/or polypeptide subunitvaccines, for example, can be used, as suggested by Walsh et al. (1987)J. Infect. Dis. 155:1198-1204 and Murphy et al. (1990) Vaccine8:497-502.

[0188] Typically, the attenuated recombinant RSV of this invention asused in a vaccine is sufficiently attenuated such that symptoms ofinfection, or at least symptoms of serious infection, will not occur inmost individuals immunized (or otherwise infected) with the attenuatedRSV. In embodiments in which viral components (e.g., the nucleic acidsor polypeptides herein) are used as vaccine or immunogenic components,serious infection is not typically an issue. In some instances, theattenuated RSV (or RSV components of the invention) can still be capableof producing symptoms of mild illness (e.g., mild upper respiratoryillness) and/or of dissemination to unvaccinated individuals. However,virulence is sufficiently abrogated such that severe lower respiratorytract infections do not typically occur in the vaccinated or incidentalhost.

[0189] While stimulation of a protective immune response with a singledose is preferred, additional dosages can be administered, by the sameor different route, to achieve the desired prophylactic effect. Inneonates and infants, for example, multiple administrations may berequired to elicit sufficient levels of immunity. Administration cancontinue at intervals throughout childhood, as necessary to maintainsufficient levels of protection against wild-type RSV infection.Similarly, adults who are particularly susceptible to repeated orserious RSV infection, such as, for example, health care workers, daycare workers, family members of young children, the elderly, andindividuals with compromised cardiopulmonary function may requiremultiple immunizations to establish and/or maintain protective immuneresponses. Levels of induced immunity can be monitored, for example, bymeasuring amounts of neutralizing secretory and serum antibodies, anddosages adjusted or vaccinations repeated as necessary to elicit andmaintain desired levels of protection.

[0190] Alternatively, an immune response can be stimulated by ex vivo orin vivo targeting of dendritic cells with virus. For example,proliferating dendritic cells are exposed to viruses in a sufficientamount and for a sufficient period of time to permit capture of the RSVantigens by the dendritic cells. The cells are then transferred into asubject to be vaccinated by standard intravenous transplantationmethods.

[0191] Optionally, the formulation for prophylactic administration ofthe RSV also contains one or more adjuvants for enhancing the immuneresponse to the RSV antigens. Suitable adjuvants include, for example:complete Freund's adjuvant, incomplete Freund's adjuvant, saponin,mineral gels such as aluminum hydroxide, surface active substances suchas lysolecithin, pluronic polyols, polyanions, peptides, oil orhydrocarbon emulsions, bacille Calmette-Guerin (BCG), Corynebacteriumparvum, and the synthetic adjuvant QS-21.

[0192] If desired, prophylactic vaccine administration of RSV can beperformed in conjunction with administration of one or moreimmunostimulatory molecules. Immunostimulatory molecules include variouscytokines, lymphokines and chemokines with immunostimulatory,immunopotentiating, and pro-inflammatory activities, such asinterleukins (e.g., IL-1, IL-2, IL-3, IL-4, IL-12, IL-13); growthfactors (e.g., granulocyte-macrophage (GM)-colony stimulating factor(CSF)); and other immunostimulatory molecules, such as macrophageinflammatory factor, Flt3 ligand, B7.1; B7.2, etc. The immunostimulatorymolecules can be administered in the same formulation as the RSV, or canbe administered separately. Either the protein or an expression vectorencoding the protein can be administered to produce an immunostimulatoryeffect.

[0193] Although vaccination of an individual with an attenuated RSV of aparticular strain of a particular subgroup can induce cross-protectionagainst RSV of different strains and/or subgroups, cross-protection canbe enhanced, if desired, by vaccinating the individual with attenuatedRSV from at least two strains, e.g., each of which represents adifferent subgroup. Similarly, the attenuated RSV vaccines of thisinvention can optionally be combined with vaccines that induceprotective immune responses against other infectious agents.

[0194] Kits and Reagents

[0195] The present invention is optionally provided to a user as a kit.For example, a kit of the invention contains one or more nucleic acid,polypeptide, antibody, or cell line described herein. Most often, thekit contains a diagnostic nucleic acid or polypeptide (e.g., an antibodyor a probe, e.g., as a cDNA microarray packaged in a suitable container)or other nucleic acid such as one or more expression vector. The kittypically further comprises one or more additional reagents, e.g.,substrates, labels, primers, tubes and/or other accessories, reagentsfor collecting samples, buffers, hybridization chambers, cover slips,etc. The kit optionally further comprises an instruction set or usermanual detailing preferred methods of using the kit components.

[0196] Digital Systems

[0197] The present invention provides digital systems, e.g., computers,computer readable media and integrated systems comprising characterstrings corresponding to the sequence information herein for thepolypeptides and nucleic acids herein, including, e.g., those sequenceslisted herein and the various silent substitutions and conservativesubstitutions thereof. Integrated systems can further include, e.g.,gene synthesis equipment for making genes and/or peptide synthesisequipment for making polypeptides corresponding to the characterstrings.

[0198] Various methods known in the art can be used to detect homologyor similarity between different character strings, or can be used toperform other desirable functions such as to control output files,provide the basis for making presentations of information including thesequences and the like. Examples include BLAST, discussed supra.Computer systems of the invention can include such programs, e.g., inconjunction with one or more data file or data base comprising asequence as noted herein.

[0199] Thus, different types of homology and similarity of variousstringency and length can be detected and recognized in the integratedsystems herein. For example, many homology determination methods havebeen designed for comparative analysis of sequences of biopolymers, forspell-checking in word processing, and for data retrieval from variousdatabases. With an understanding of double-helix pair-wise complementinteractions among 4 principal nucleobases in natural polynucleotides,models that simulate annealing of complementary homologouspolynucleotide strings can also be used as a foundation of sequencealignment or other operations typically performed on the characterstrings corresponding to the sequences herein (e.g., word-processingmanipulations, construction of figures comprising sequence orsubsequence character strings, output tables, etc.).

[0200] Thus, standard desktop applications such as word processingsoftware (e.g., Microsoft Word™ or Corel WordPerfec™) and databasesoftware (e.g., spreadsheet software such as Microsoft Excel™, CorelQuattro Pro™, or database programs such as Microsoft Access™or Paradox™)can be adapted to the present invention by inputting a character stringcorresponding to one or more polynucleotides and polypeptides of theinvention (either nucleic acids or proteins, or both). For example, asystem of the invention can include the foregoing software having theappropriate character string information, e.g., used in conjunction witha user interface (e.g., a GUI in a standard operating system such as aWindows, Macintosh or LINJX system) to manipulate strings of characterscorresponding to the sequences herein. As noted, specialized alignmentprograms such as BLAST can also be incorporated into the systems of theinvention for alignment of nucleic acids or proteins (or correspondingcharacter strings).

[0201] Systems in the present invention typically include a digitalcomputer with data sets entered into the software system comprising anyof the sequences herein. The computer can be, e.g., a PC (Intel x86 orPentium chip- compatible DOS™, OS2™ WIDOWS™ WINDOWS NT™, WINDOWS95™,WINDOWS98™ LINUX based machine, a MACINTOSH™, Power PC, or a UNIX based(e.g., SUN™ work station) machine) or other commercially common computerwhich is known to one of skill. Software for aligning or otherwisemanipulating sequences is available, or can easily be constructed by oneof skill using a standard programming language such as Visualbasic,Fortran, Basic, Java, or the like.

[0202] Any controller or computer optionally includes a monitor which isoften a cathode ray tube (“CRT”) display, a flat panel display (e.g.,active matrix liquid crystal display, liquid crystal display), orothers. Computer circuitry is often placed in a box which includesnumerous integrated circuit chips, such as a microprocessor, memory,interface circuits, and others. The box also optionally includes a harddisk drive, a floppy disk drive, a high capacity removable drive such asa writeable CD-ROM, and other common peripheral elements. Inputtingdevices such as a keyboard or mouse optionally provide for input from auser and for user selection of sequences to be compared or otherwisemanipulated in the relevant computer system.

[0203] The computer typically includes appropriate software forreceiving user instructions, either in the form of user input into a setparameter fields, e.g., in a GUI, or in the form of preprogrammedinstructions, e.g., preprogrammed for a variety of different specificoperations. The software then converts these instructions to appropriatelanguage, e.g., for instructing the operation of equipment, e.g., geneand/or peptide synthesis equipment, to carry out the desired operation.

[0204] The software can also include output elements for controllingnucleic acid synthesis (e.g., based upon a sequence or an alignment of asequences herein) or other operations.

[0205] In an additional aspect, the present invention provides systemkits embodying the methods, composition, systems and apparatus herein.System kits of the invention optionally comprise one or more of thefollowing: (1) an apparatus, system, system component or apparatuscomponent as described herein; (2) instructions for practicing themethods described herein, and/or for operating the apparatus orapparatus components herein and/or for using the compositions herein. Ina further aspect, the present invention provides for the use of anyapparatus, apparatus component, composition or kit herein, for thepractice of any method or assay herein, and/or for the use of anyapparatus or kit to practice any assay or method herein.

EXAMPLES

[0206] The following sets forth a series of experiments that demonstrateconstruction of RSV B9320 cDNAs and recovery of recombinant RSV from thecDNAs. It is understood that the examples and embodiments describedherein are for illustrative purposes only and that various modificationsor changes in light thereof will be suggested to persons skilled in theart and are to be included within the spirit and purview of thisapplication and scope of the appended claims. Accordingly, the followingexamples are offered to illustrate, but not to limit, the claimedinvention.

Materials and Methods

[0207] Cells and Viruses

[0208] Monolayer cultures of HEp-2 and Vero cells (obtained from theAmerican Type Culture Collection, ATCC) were maintained in minimalessential medium (MEM) containing 5% fetal bovine serum. RSV A2 wasobtained from the ATCC and grown in Vero cells in Opti-MEM. RSV subgroupB strain 9320, originally isolated in Massachusetts in 1977 (Hierholzerand Hirsch (1979) J. Infect. Dis. 140:826-828), was obtained from theATCC and grown in Vero cells in Opti-MEM. Infected cells were maintainedin serum-free Opti-MEM medium. Modified vaccinia virus Ankara expressingbacteriophage T7 RNA polymerase (MVA-T7; Sutter et al. (1995) FEBS Lett.371:9-12 and Wyatt et al. (1995) Virology 210:202-205) was provided byDr. Bernard Moss and amplified in CEK cells. Recombinant fowlpox virusexpressing the T7 RNA polymerase (FPV-T7; Britton et al. (1996)“Expression of bacteriophage T7 RNA polymerase in avian and mammaliancells by a recombinant fowlpox virus” J Gen Virol 77:963-7) was obtainedfrom Dr. Michael Skinner and grown in CEK cells.

[0209] Sequencing

[0210] RSV B9320 was grown in Vero cells and viral RNA was extractedfrom virus purified by ultracentrifugation from infected cell culturesupernatant. 9320 genome sequences were obtained by sequencing DNAfragments generated by RT-PCR; the cDNA full length clone was alsosequenced for comparison. All sequencing was done by Sequetech, MountainView Calif. (www.sequetech.com).

[0211] Sequence Analysis

[0212] Sequence analysis was performed with Vector NTI version 6.0 and8.0 ContigExpress and AlignX (Informax, Inc., www.informaxinc.com).Pairwise nucleic acid or polypeptide sequence alignments were performedwith Vector NTI AlignX using default parameters set by the provider.

[0213] Construction of Full-length cDNA of RSV Subgroup B9320 Strain andRecovery of Infectious Respiratory Syncytial Virus from cDNA

[0214] Construction of RSV 9320 Protein Expression Plasmids

[0215] The 9320 N, P, and L protein coding regions were each cloned intoa pCITE vector (Novagen, Madison, Wis.) under control of a T7 RNApolymerase promoter, to produce expression plasmids pB-N, pB-P, andpB-L.

[0216] pB-N: The N gene was amplified by RT-PCR from 9320 RNA extractedfrom virus particles purified by ultracentrifugation, using primers XC19(5′-GATCCCATGGCTCTTAGCAAAGTCAAG-3′ containing Nco I site, SEQ ID NO:20)and XC020 (5′-GTACGGATCCGTTGACTTATTTGCCCCGTAT-3′ containing BamHI site,SEQ ID NO:21), and cloned between the NcoI and BamHI sites of pCITE2a/3a(Novagen) under the control of T7 promoter. This N protein expressionplasmid was designated as AD740.

[0217] pB-P: The P gene was amplified by RT-PCR using primers XC17(5′-GATCCCATGGAGAAGTTTTGCACCTG-3′ with Nco I site, SEQ ID NO:22) andXC018 (5′-GTACGGATCCTGAGTGAGTTGATCACTG-3′ with BamH I site, SEQ IDNO:23) and cloned between the NcoI and BamHI sites of pCITE2a/3a. Thisclone was designated as AD741.

[0218] pB-L: The L gene was cloned from three cDNA subclones obtained byRT-PCR and the clone was assigned as AD778.

[0219] To obtain one subclone, primers XC0O3 (5′GCTTGGCCATAACGATTCTATATCATCC-3′, SEQ ID NO:24) and XC014(5′-GGTAGTATAATGTTGTGCACTTTAG-3′, SEQ ID NO:25) were used to amplify9320 L from nt8511 to nt11685 and the cDNA was cloned into T/A vector(Invitrogen, pCR 2.1) to generate subclone AD762.

[0220] To obtain the second subclone, primers XC011(5′-GGTCACGATTTACAAGATAAGCTCC-3′, SEQ ID NO:26) and XC007(5′-CAGATCCTTTTAACTTGCTACCTAGGCACA-3′ SEQ ID NO:27) were used to amplifynt 11686 to nt14495 and the BamH I to Avr II fragment was cloned intothe T/A vector as AD763.

[0221] To obtain the third subclone, primers XC009(5′-CTTACGTGTGCCTAGGTAGCAAG-3′, SEQ ID NO:28) and XC010(5′-ACGAGAAAAAAAGTGTCAAAAACTAATGTCTCG, SEQ ID NO:29) were used toamplify 9320 nt 14495 to 15225, producing a first PCR product. To addthe ribozyme cleavage sequence (RBZ) and T7 terminator sequence (T7ø), asecond PCR product was obtained using XC015(5′-GTTTTTGACACTTTTTTTCTCGTGGCCGGCATGGTCCCAGCC-3′, SEQ ID NO:30) andXC016 (5′-GATCTAGAGCTCCAAGCTTGCGGCCGCGTCGAC-3′ containing the Kpn Isite, SEQ ID NO:31) as primers and pRSV-A2 full-length antigenomic cDNA(Jin et al. (1998) Virology 251:206-214) as template. Since primersXC010 and XC015 contained overlapping sequences, these two PCR productswere annealed, extended and amplified by PCR using XC009 and XC016 asprimers. The cDNA was cloned into the T/A vector and designated asAD764.

[0222] The three subclones were verified by sequence analysis. To jointhe three L subclones together, the Avrll and Kpn I fragment was removedfrom AD764 and cloned into AD763 and the larger clone was designated asAD767. The BamHI to NotI restriction fragment from nt11685 to thesequence downstream of the T7 terminator was removed from AD767 andcloned into pCITE2a/3a vector under the control of the T7 promoter andthe plasmid was designated as AD766. The BamHI fragment from nt 8511 tont 11685 was removed from clone AD762 and inserted into the BamH I siteof AD766. The second BamHI site at position of nt 11685 was then knockedout by site-directed mutagenesis and the clone is designated as pB-L(AD778).

[0223] The functions of the pB-N, pB-P, and pB-L expression plasmidswere examined by the RSV minigenome assay (as described in, e.g., Tanget al. (2002) Virology 302:207-216). A level of the CAT reporter genesimilar to those of A2 expression plasmids was detected in cellstransfected with pB-N, pB-P, pB-L and pRSVCAT minigenome, indicating allthree of these plasmids are functional.

[0224] Assembly of Full-length Antigenomic cDNA of RSV 9320 Strain

[0225] An antigenomic cDNA spanning the entire RSV 9320 genome wasassembled by sequential ligation of RSV cDNA fragments with theindicated unique restriction sites (FIG. 1). In brief, six cDNAfragments (B1-B6) were generated from 9320 viral RNA by RT-PCR using thepfu polymerase (Stratagene, La Jolla, Calif.) and cloned into a modifiedpET vector containing the RSV 9320 unique restriction enzyme sites (XmaI, Avr II, Sac I, BamH I, and BssHII). The Xma I-Avr II cDNA fragment(B1) containing the T7 RNA polymerase promoter proximal to the 5′antigenomic sense DNA was joined with the Avr II-Sac I cDNA fragment(B2) to form the B7 fragment (through a SacI site, as described below).The B7 fragment was used to replace the corresponding region in afull-length RSV A2-B9320 chimera containing the G, F and M2 sequence(B3). The Sac I restriction site at nt 2310 in the resulting pUC-B8 wasmutated without affecting the coding sequence of the SH gene. The L genefragment (B10) was assembled from B4 and B9 fragments. The hepatitisdelta virus ribozyme (RBZ) and the T7 RNA polymerase terminator sequencewas amplified from RSV A2 antigenomic cDNA (Jin et al. (1998)“Recombinant human respiratory syncytial virus (RSV) from cDNA andconstruction of subgroup A and B chimeric RSV” Virology 251:206-214) andligated to the trailer sequence through PCR (B6). Ligation of B5 and B6fragments were mediated through the Avr II and Not I restriction sites.The BamH I site at nt position 11685 was deleted from B10 cDNA bymutagenesis without affecting the protein coding sequence. The L gene(B10) was cloned into the chimeric clone that contained the 9320 B8fragment and A2 L to replace the A2-L sequence through the BamH I andNot I restriction sites. The antigenomic cDNA clone (B11) encoding thecomplete RSV 9320 genome was designated pB9320C4. In addition, anantigenomic cDNA clone containing a single C to G change at the fourthposition of the leader sequence was also obtained by mutagenesis anddesignated pB9320G4. A more detailed description of the cDNAconstruction follows.

[0226] Positions of various primers and subclones used in cloning thefull length cDNA are illustrated in FIGS. 1-3. The 3′ genome wasamplified by RT-PCR using PFU polymerase with primer V1964(5′-GGGTACCCCCGGGTAATACGACTCACTATAGGGACGGGAAAAAATG-3′ containing Xma Irestriction enzyme, the T7 promoter and the leader sequences, SEQ IDNO:32) and XC051 (5′-GTTAACTTAGAGCTCTACATCATC-3′ containing the Sac Irestriction site present in 9320 genome at position of nt 2310, SEQ IDNO:33). The PCR fragment was cloned between the Xma I and SacI sites ofthe modified pET vector (pET21b was cut with BspEI, and the pET21b wasligated with a polylinker with XmaI, SmaI, SacI, MscI, BamHI, SpeI,PmII, and BssHII restriction sites to produce the modified pET-21bvector) and designated as AD803. A second PCR DNA that contained nt 2106to 4494 was obtained by RT/PCR using XC034(5′-GTGTGGTCCTAGGCAATGCAGCAG-3′, SEQ ID NO:34) and XC032(5′-GACACAGCATGATGGTAGAGCTCTATGTG-3′, SEQ ID NO:35) as primers and wascloned into the Sac I site of the pET vector for sequence analysis. ThisSacI fragment was then moved to AD803 through the Sac I site in AD803(producing B7). In order to ligate the cDNA encoding NS1, NS2, N, P, Mand SH genes with the rest of the 9320 cDNA, the Sac I site at positionnt 2310 was removed by mutagenesis and the resulting clone wasdesignated as AD816. The Xma I to Sac I fragment from AD816 was releasedto replace the corresponding region of the A2 sequence in the chimericcDNA AD379 that had the G and F genes of 9320 in place of A2 G and Fgenes (in a pUC19 backbone, Cheng et al. (2001) Virology 283:59-68).This clone was designated as AD827.

[0227] The cDNA containing the G, F, and M2 genes of 9320 strain wasderived by RT-PCR using XC063 (5′-GCTAAGTGAACATAAAACATTCTGTAAC-3′, SEQID NO:36) as RT primer and XC006 (5′-CCATTAATAATGGGATCCATTTTGTC-3′ withSacI site, SEQ ID NO:37) and XC062 (5′-CACATAGAGCTCTACCATCATGCTGTGTC-3′with BamH I site, SEQ ID NO:38) as PCR primers and was initially clonedinto pET vector as AD835 for sequence analysis. The SacI to BamH Ifragment from AD835 was then moved to AD827 and the clone was designatedas AD848.

[0228] The BamHI and NotI fragment from pB-L (AD869 encoding 9320 L withthe two Sac I sites at positions of nt 10376 and nt 14951 knocked-out)was swapped into AD848 to complete the assembly of a full lengthantigenomic cDNA of RSV 9320.

[0229] Three mutations introduced by PCR during the cloning process werecorrected by site-directed mutagenesis in their respective subclones, asfollows. To reverse a His to Asn change in L at amino acid position 209,site-directed mutagenesis was performed to correct the His with primersXC081 (5′-CATGGTTAATACACTGGTTCAATTTATATACA-3′, SEQ ID NO:41) and XC082(5′-TGTATATAAATTGAACCAGTGTATTAACCATG-3′, SEQ ID NO:42). To correct anArg to Lys change in N at amino acid position 194 (nt 1748), sitedirected mutagenesis was performed with primers XC086(5′-GTCTTAAAAAACGAAATAAAACGCTACAAGGGCCTCATACC-3′, SEQ ID NO:43) andXC087 (5′GGTATGAGGCCCTTGTAGCGTTTTATTTCGTTTTTTAAGAC-3′, SEQ ID NO:44). ASer to Asn change in NS1 at amino acid position 108 was not corrected.

[0230] The recombinant 9320 cDNA has the following genetic tags that aredifferent from wild-type 9320 virus. (The enclosed sequence, SEQ IDNO:1, is the wild type RSV 9320 strain and does not reflect therecombinant DNA sequence.) First, Sac I sites at nt 2310, 10376 and14951 were removed without changing the protein coding sequences, usingthe following primers: SacI at 2310 nt, XC049(5′-GATGATGTAGAGCTTTAAGTTAAC-3′, SEQ ID NO:45) and XC050(5′-GTTAACTTAAAGCTCTACATCATC-3′, SEQ ID NO:46); SacI at 10376 nt, XC088(5′-CTAACTGGTAAAGAAAGAGAGCTTAGTGTAGGTAGAATGTTTGC-3′, SEQ ID NO: 47) andXC089 (5′-GCAAACATTCTACCTACACTAAGCTCTCTTTCTTTACCAGTTAG-3′, SEQ ID NO:48); and SacI at 14951 nt, XC090(5′-GTTTAACAACCAATGAGCTTAAAAAGCTGATTAAAATTAC-3′, SEQ ID NO:49) and XC091(5′-GTAATTTTAATCAGCTTTTAAGCTCATTGGTTGTTAAAC-3′, SEQ ID NO:50). Theremoval of the two Sac I sites at positions nt 10376 and 14951 in the Lgene from AD864 to generate AD869 did not alter the amino acid sequenceof L. Second, a BamH I site at nt 11685 was removed using primers XC067(5′-CATTAATGAGGGACCCACAGGCTTTAG-3′, SEQ ID NO:39) and XC068(5′-CTAAAGCCTGTGGGTCCCTCATTAATG-3′, SEQ ID NO:40). Third, a Sac I siteat nt 4477 was added using XC032 (5′-GACACAGCATGATGGTAGAGCTCTATGTG, SEQID NO:35).

[0231] It was previously reported that changing the C at the fourthnucleotide position of the leader region of RSV A2 to a G increased thepromoter strength, resulting in increased transcription/replication ofan RSV minigenome (Collins et al. (1993) “Rescue of a 7502-nucleotide(49.3% of full-length) synthetic analog of respiratory syncytial virusgenomic RNA” Virology 195:252-256) and higher virus recovery efficiency(Jin et al. (1998) Virology 251:206-214). Thus, as noted, a 9320 cDNAwith a C4 to G change in the leader sequence at the antigenomic sensewas made to increase the promoter strength, and the resulting clone wasdesignated as AD897 (pB9320G4).

[0232] Construction of G Gene Deletion Mutants

[0233] Two mutants were constructed to determine if the G gene of RSVB9320 strain is dispensable, e.g., for viral replication in tissueculture and/or an animal host. In one mutant, the entire open readingframe of the G gene was removed from the 9320 cDNA. In the other mutant,the region encoding the cysteine noose and heparin binding sites of Gwas removed from the 9320 cDNA.

[0234] For the RSV A2 strain, the G protein has been shown to bedispensable for virus replication in vitro (Techaarpomkul et al. (2001)“Functional analysis of recombinant respiratory syncytial virus deletionmutants lacking the small hydrophobic and/or attachment glycoproteingene” J Virol 75:6825-34 and Teng et al. (2001) “Contribution of therespiratory syncytial virus G glycoprotein and its secreted andmembrane-bound forms to virus replication in vitro and in vivo” Virology289:283-96). However, rA2ΔG (RSV A2 lacking G) replicated poorly inHEp-2 cells, and its replication was attenuated in the respiratorytracts of mice. Previously, it was also shown that a cold-adapted RSV B1strain, cp-52, had both the SH and G genes deleted (Karron et al. (1997)Proc Natl Acad Sci USA 94:13961-13966). However, this deletion mutantreplicated poorly and was over-attenuated in animals and in humans. Todetermine whether the G protein was also dispensable for 9320replication in vitro, an antigenomic cDNA was constructed in which theentire G gene (957 nt) including the gene start and gene end sequenceswas deleted from the cDNA and the new SH-F intergenic region contained75 nt.

[0235] To construct the 9320 antigenomic cDNA that had the G genedeleted, deletion mutagenesis was performed on a pET-S/B cDNA subclonethat contained sequences of the 9320 G, F and M2 genes using a pair ofPCR primers flanking the G open reading frame in opposite orientations(5′-GATCCCATACTAATAATTCATCATTATG-3′, SEQ ID NO:51, and5′-AGCAGAGAACCGTGATCTATCAAGCAAG-3′, SEQ ID NO:52) using the ExSitePCR-based Site-Directed Mutagenesis Kit (Stratagene, La Jolla, Calif.).The deletion was confirmed by restriction enzyme digestion andnucleotide sequencing analysis. The Sac I-BamH I fragment containingonly the F and M2 genes was introduced into pB9320G4, and theantigenomic cDNA was designated pB9320ΔG.

[0236] To delete only the cysteine noose and heparin binding sites of G(amino acids 164-197), a small cDNA fragment of nt 5179-5280 nt wasdeleted from the 9320 G gene using primers XC079(5′-GTAATCATCTTTTGGTTTTTTTGGTGG-3′, SEQ ID NO:53) and XC080(5′-CCAACCATCAAACCCACAAACAAACCAACCGTC-3′, SEQ ID NO:54). The cDNAcontaining the desired deletion was then removed from the subclone bydigestion with Sac I and BamHI and shuffled into the full-length 9320antigenomic cDNA. The resulting antigenomic cDNA was designatedpB9320ΔHBS.

[0237] Recovery of Recombinant Viruses from cDNAs

[0238] Recovery of 9320 viruses (rg9320C4, rg9320G4, rg9320ΔG andrg9320ΔHBS) by reverse genetics (rg) was performed as describedpreviously (Jin et al. (1998) Virology 251:206-214). Briefly, HEp-2cells were infected with MVA-T7 at an m.o.i. of 5.0 and transfected with0.4 μg of pB-N (pN), 0.4 μg of pB-P (pP), 0.2 μg of pB-L (pL), and 0.8μg of pB9320C4, pB9320G4, pB9320ΔG or pB9320ΔHBS by Lipofectamine™ 2000(Invitrogen, Carlsbad, Calif.). In some transfection reactions, 0.2 μgpRSV-M2-1 (encoding the RSV A2 M2-1 protein) was also included.Alternatively, Vero cells were infected with FPV-T7 (Britton et al.(1996) J Gen Virol 77:963-7) at an m.o.i. of 1.0 for 1 hr andtransfected with the DNAs as above. Transfected cells were incubated at35° C. Three days after transfection, the culture supernatant was usedto infect Vero cells to amplify the recovered viruses. Six days afterinfection, the culture supernatant was harvested and virus-infectedcells were identified by immunostaining using polyclonal anti-RSV A2serum (Biogenesis, Kingston, N.H.). The recombinant virus from theculture supernatant was plaque purified and amplified in Vero cells.

[0239] Replication of rg9320C4, rg9320G4, rg9320ΔG in Tissue Culture

[0240] Replication of rg9320C4, rg9320G4 and rg9320ΔG in Vero and HEp-2cells was compared with replication of wild type 9320. Vero or HEp-2cell monolayers in 6-well plates were infected with each virus induplicate at an m.o.i. of 0.1. After 1 hr adsorption at roomtemperature, the infected cells were washed with PBS three times andincubated with 2 ml of OptiMEM at 35° C. At 24 hr intervals, aliquots of250 μl of culture supernatant were removed and stored at −80° C. priorto virus titration. Each aliquot taken was replaced with the same amountof fresh media. The virus titer was determined by plaque assay on Verocells using an overlay consisting of 1% methylcellulose and 1×MEM/L15(JRH Bioscience, Lenexa, Kans.) containing 2% FBS. After incubation at35° C. for 6 days, the monolayers were fixed with methanol and plaquesenumerated by immunostaining.

[0241] Western Blotting Analysis of Virus Infected Cells

[0242] Vero or HEp-2 cells were infected with virus at an m.o.i of 5.0and the infected cells were lysed in Laemmli protein sample buffer(Bio-Rad, Hercules, Calif.). The cell lysates were electrophoresed on12% polyacrylamide gels containing 0.1% SDS, and then transferred to anylon membrane. The blots were incubated with either polyclonal anti-RSVA2 serum or a mixture of four monoclonal antibodies against the Gprotein of RSV B strain (2434DB3, 2218BD5, 2218AE7 and 2218DG7) obtainedfrom Dr. Gregory Storch (Storch et al. (1991) “Antigenic and genomicdiversity within group A respiratory syncytial virus” J Infect Dis163:858-861). Viral proteins were visualized by incubation withhorseradish peroxidase (HRP)-conjugated secondary antibodies followed bychemiluminescent detection (Amersham Biosciences, Piscataway, N.J.).

Results and Discussion

[0243] We have described the construction of a full-length antigenomiccDNA derived from RSV subgroup B9320 strain and recovery of infectiousvirus from the cDNA. The antigenomic sequence (complementary to thewild-type RSV 9320 genome and not including the changes introduced intoour recombinant RSV) is listed as SEQ ID NO:1. The sequence is beingdeposited in GenBank (accession number AY353550).

[0244] The RSV 9320 genome contains 15,225 nucleotides and shares 97.8%and 86% identity compared to RSV B1 and A2 strains, respectively. Asnoted previously, the A2 genome contains 15,222 nucleotides; the B1genome contains 15,225 nucleotides (Karron et al. (1997) Proc Natl AcadSci USA 94:13961-13966). Like the RSV A2 strain, 9320 contains 10transcriptional units encoding 11 proteins in the order ofNS1/NS2/N/P/M/SH/G/F/M2-1/M2-2/L. Amino acid sequences of the proteinsare also provided: NS1 is listed as SEQ ID NO:2, NS2 as SEQ ID NO:3, Nas SEQ ID NO:4, P as SEQ ID NO:5, M as SEQ ID NO:6, SH as SEQ ID NO:7, Gas SEQ ID NO:12, F as SEQ ID NO:8, M2-1 as SEQ ID NO:9, M2-2 as SEQ IDNO:10, and L as SEQ ID NO:11.

[0245] Table 3 lists the size of each of the 11 proteins for the 9320,B1, and A2 strains. TABLE 3 Length of protein (amino acids) B9320 B 1 A2NS 1 139 139 139 NS 2 124 124 124 N 391 391 391 P 241 241 241 M 256 256256 SH 65 65 64 G 292 299 298 F 574 574 574 M2-1 195 195 194 M2-2 93 9390 L 2166 2166 2165

[0246] Table 4 lists the length of the intergenic regions for the threeRSV strains. TABLE 4 Length of intergenic region (nucleotides)INTERGENIC B9320 B 1 A2 NS 1/NS 2 16 16 19 NS 2/N 23 23 26 N/P 3 3 1 P/M9 9 9 M/SH 9 9 9 SH/G 44 44 44 G/F 52 52 52 F/M2 56 56 46 GS L/GE M2 4646 46

[0247] Table 5 lists the percentage amino acid sequence identity betweenstrains B9320 and B1, 9320 and A2, and A2 and B1 for each protein. TheSH, G and M2-2 proteins display the greatest differences between A2 and9320, while the other proteins have an amino acid identity greater than86%. TABLE 5 Amino acid identity (%) RSV Gene B9320/B1 B9320/A2 A2/B1 NS1 99.3 86.3 87 NS 2 98.4 90.4 92 N 99.7 95.9 96 P 98.3 90.9 91 M 99.692.2 91 SH 97.0 71.2 76 G 90.3 52.2 53 F 99.3 89.2 89 M2-1 99.5 92.8 N/AM2-2 96.4 62.4 92 L 99.2 92.4 93

[0248] Recovery of Infectious 9320 from cDNA

[0249] A reverse genetics system for the A2 strain of subgroup A RSV wasestablished several years ago (Collins et al. (1995) Proc. Natl. Acad.Sci. USA 92:11563-11567 and Jin et al. (1998) Virology 251:206-214).However, a system for recovery of subgroup B RSV has not previously beenavailable. Herein we describe construction of an antigenomic cDNAderived from RSV subgroup B9320 and recovery of infectious RSV fromcDNA. Similar to the A2 strain, rescue of 9320 depends on the expressionof the viral polymerase proteins N, P and L. The M2-1 expression plasmidis not required for RSV 9320 recovery in either the FPV-T7 infected Veroor the MVA-T7 infected HEp-2 cells. However, M2-1 function was probablysupplied by cryptic expression of the M2-1 protein from the transfectedfull-length antigenomic cDNA (Collins et al. (1999) Virology259:251-255). The establishment of the reverse genetics system for the9320 strain should greatly aid studies of viral protein structure andfunction of this divergent RSV subgroup.

[0250] Recovery of infectious RSV requires co-transfection of a minimumof three plasmids encoding the N, P and L proteins (Jin et al. (1998)Virology 251:206-214). In addition, the elongation function of M2-1 isalso required for the virus recovery from cDNA (Collins et al. (1999)“Support plasmids and support proteins required for recovery ofrecombinant respiratory syncytial virus” Virology 259:251-255 andCollins et al. (1995) “Production of infectious human respiratorysyncytial virus from cloned cDNA confirms an essential role for thetranscription elongation factor from the 5′ proximal open reading frameof the M2 mRNA in gene expression and provides a capability for vaccinedevelopment” Proc. Natl. Acad. Sci. USA 92:11563-11567). The expressionplasmids encoding the 9320 N, P, and L proteins were constructed andtheir functions were examined in the RSV minigenome assay using thepRSV/CAT replicon that contained the negative sense CAT gene flanked bythe leader and trailer sequences derived from RSV A2 strain (Tang et al.(2001) J Virol 75:11328-11335). The minigenome assay indicated that the9320 N, P and L expression plasmids functioned as well as those of RSVA2 strain.

[0251] To recover virus from RSV 9320 cDNA, pB9320C4 or pB9320G4 wastransfected into MVA-T7 infected BEp-2 cells or FPV-T7 infected Verocells together with the 9320 N, P and L expression plasmids with orwithout the RSV A2 M2-1 plasmid. Several days after inoculation of Verocells with the transfected culture supernatant, syncytia formation wasobserved in the infected Vero cells and virus infection was confirmed byimmunostaining.

[0252] Table 6 lists the recovery efficiency of recombinant RSV in thepresence or absence of the M2-1 expression plasmid. FPV-T7 infected Verocells or MVA-T7 infected HEp-2 cells were transfected with p9320C4 orp9320G4 in triplicate wells together with N, P, L expression plasmidswith or without M2-1. Three days after transfection, the culturesupernatants were titrated on Vero cells and the plaque numbers inpfu/ml from each well are shown. The average plaque number is given inparentheses. TABLE 6 Average plague number (pfu/ml) MVA-T7 infectedFPV-T7 infected Vero cells (pfu/ml) HEp-2 cells (pfu/ml) Virus +M2-1−M2-1 +M2-1 −M2-1 rg932004 70, 210, 93 (124) 40, 40, 75 (52) 55, 5, 3(21) 18, 0, 0 (6) rg9320G4 1075, 1860, 1280 (1405) 1140, 890, 900 (977)285, 3, 0 (96) 18, 28, 0 (15)

[0253] As shown in Table 6, rg9320G4 was rescued more efficiently thanrg9320 in both the FPV-T7 infected Vero cells and the MVA-T7 infectedHEp-2 cells. Inclusion of the M2-1 expression plasmid slightly increasedrescue efficiency in both cell types.

[0254] For vaccine production, the vaccine is typically produced from aqualified cell line that is free of any adventitious agents. The BEp-2cells currently used for recovery of infectious RSV A2 (e.g., Collins etal. (1995) Proc. Natl. Acad. Sci. USA 92:11563-11567 and Jin et al.(1998) Virology 251:206-214) are not suitable for vaccine production.Vero cells were therefore explored as the cell substrate for recovering9320 virus from its cDNA. It was very difficult to recover virus fromMVA-T7-infected Vero cells. FPV-T7 has been shown to have a lesscytopathic effect in infected Vero cells and thus result in moreefficient virus rescue of other viruses (Britton et al. (1996) J GenVirol 77:963-7 and Das et al. (2000) “Improved technique for transientexpression and negative strand virus rescue using fowlpox T7 recombinantvirus in mammalian cells” J Virol Meth 89:119-127). Similarly, therecombinant RSV described here were more efficiently recovered fromFPV-T7 infected Vero cells than from MVA-T7 infected cells, which maypave the way for recovering RSV vaccine candidates for clinical studies.

[0255] rg9320C4 and rg9320G4 were plaque purified and amplified in Verocells, both reaching a titer of 1×10⁷ pfu/ml. A recombinant virus withthe G gene deleted from rg9320G4 was also obtained and the virus wasdesignated rg9320ΔG.

[0256] The identity of the recombinant viruses generated from cDNA wasanalyzed by RT/PCR of each viral RNA using primer pairs spanning theintroduced marker sites or the G deletion region. Digestion of theRT/PCR DNA product from nt 2104 to nt 3096 by the Sac I restrictionenzyme showed that the Sac I site was present in 9320 virus but not inthe recombinant viruses rg9320C4 and rg9320ΔG. Digestion of the RT/PCRproduct from nt 11593 to nt 11822 by BamH I confirmed that the BamH Isite in the recombinant viruses was also abolished. RT/PCR using a pairof primers spanning the G gene confirmed that the G gene was deletedfrom rg9320ΔG, since its DNA product was approximately 1 kb shorter thanthat of rg9320. Sequencing of the G deletion junction region confirmedthe expected deleted sequence.

[0257] The lack of G protein expression in rg9320ΔG infected cells wasalso confirmed by Western blotting analysis using monoclonal antibodiesagainst the G protein of subgroup B RSV (Storch et al. (1991) J InfectDis 163:858-861). The G protein was not expressed in rg9320ΔG-infectedcells. Western blotting using a polyclonal antibody against RSV revealedan equivalent level of other viral proteins (F, N and P) synthesized byrg9320ΔG compared with rg9320G4.

[0258] Replication of the G Deletion Mutant in Tissue Culture

[0259] Replication of recombinant 9320 and its G deletion mutant werecompared with replication of the biologically derived 9320 strain. Veroor HEp-2 cells were infected with 9320, rg9320C4, rg9320G4 or rg9320ΔGat an m.o.i. of 0.1, and the accumulated level of viruses released intothe culture supernatant at each day was titrated in Vero cells. As shownin FIG. 4 Panel A, the growth kinetics of rg9320C4, rg9320G4 andrg9320ΔG were very similar to those of 9320 in Vero cells, reaching peaktiters of 6.0 log₁₀ pfu/ml at 96 hours post infection. In HEp-2 cells(FIG. 4 Panel B), rg9320C4 grew similarly to 9320 whereas rg9320G4 grewslightly slower. Replication of rg9320ΔG was reduced by 5-fold comparedto rg9320G4.

[0260] The plaque morphology of rg9320ΔG was compared to that ofrg9320G4 in Vero and HEp-2 cells. The spread of rg9320ΔG in Vero andHEp-2 cells in liquid medium was similar to that of rg9320G4. rg9320ΔGalso formed plaques of similar size in both the Vero and HEp-2 cells.These data indicated that the deletion of G from 9320 did not havesignificant impact on virus replication in Vero and Hep-2 cells.

[0261] We have demonstrated that the G protein is not essential for RSV9320 replication in tissue culture cells. However, deletion of G fromRSV A2 strain severely affected virus replication in HEp-2 cells; rA2ΔGhad a reduction of more than 3.0 log₁₀ and did not form distinct plaquesin HEp-2 cells (Teng et al. (2001) Virology 289:283-96). A study byTechaarpornkul et al. (Techaarpornkul et al. (2001) J Virol 75:6825-34)showed that rA2ΔG had 1-2 logs lower titer in HEp-2 cells and formedsmaller plaques in HEp-2 cells. The G protein enhances virus binding totarget cells, but it has no role in virus penetration once virus hasattached the cells (Techaarpornkul et al. (2001) J Virol 75:6825-34). InVero cells, rA2ΔG replicated as well as wt A2 strain, indicating analternative pathway might be present in Vero cells for efficient virusentry that is independent of the G protein (Teng et al. (2001) Virology289:283-96). Like rA2ΔG, rg9320ΔG replicated efficiently in Vero cells;however, in contrast to rA2ΔG, its replication was only slightly reducedin HEp-2 cells. The reduction of rg9320ΔG virus in HEp-2 cells was5-fold compared to the parental rg9320G4 virus and the plaque sizereduction in HEp-2 cells was also less apparent. Therefore, 9320 appearsto be less dependent on the G protein for infectivity in HEp-2 cellsthan RSV A2.

[0262] rg9320ΔHBS, a recombinant 9320 virus with a partial deletion of G(the cysteine noose and heparin binding sites) was also recovered fromcDNA as described above. Like rg9320ΔG, replication of rg9320ΔHBS inVero cells is not significantly impaired.

[0263] Virus Replication in Cotton Rats

[0264] Subgroup B RSV typically replicates better in cotton rats than inmice. Therefore, in vivo replication of recombinant viruses rg9320C4,rg9320G4, rg9320ΔG and rg9320ΔHBS was examined in cotton rats (Sigmodonhispidus). Cotton rats in groups of five were inoculated intranasallywith 150 μl of inoculum containing 10⁶ pfu of the specified virus peranimal. Animals were sacrificed four days after inoculation. Lungtissues were harvested and homogenized, and virus titer was determinedby plaque assay on Vero cells. Table 7 lists the mean viral titer foreach recombinant virus. rg9320C4 and rg9320G4 replicated to a titer of3.1 and 3.0 log₁₀ pfu/g, respectively, in the lungs of cotton rats. BothG deletion mutants (rg9320ΔG and rg9320ΔHBS) replicated poorly,indicating G deletion affected RSV 9320 replication in the animal host.rg9320ΔG and rg9320ΔHBS are thus potential candidates for liveattenuated RSV vaccines. TABLE 7 Virus replication in cotton rat lungvirus log10 mean pfu/g tissue +/− SE rg9320C4 3.1 +/− 0.2 rg9320G4 3.0+/− 0.1 rg9320ΔG 0.4 +/− 0.4 rg9320ΔHBS 1.0 +/− 0.6

[0265] Recombinant 9320 and Vaccines

[0266] An RSV vaccine would preferably provide protection against bothsubgroup A and subgroup B RSV infection, which tend to circulateconcurrently in communities. Most RSV vaccines developed in the pasthave been based on the RSV A2 strain. However, it remains to bedetermined whether an RSV vaccine based solely on a subgroup A strainwould provide sufficient immunity to both RSV subgroups. Althoughrecombinant technology has been employed to express subgroup B RSVantigens in the A2 strain, including the replacement of the G and Fgenes by those of BI (Whitehead et al. (1999) “Replacement of the F andG proteins of respiratory syncytial virus (RSV) subgroup A with those ofsubgroup B generates chimeric live attenuated RSV subgroup B vaccinecandidates” J Virol 73:9773-9780) or 9320 (Cheng et al. (2001) “Chimericsubgroup A respiratory syncytial virus with the glycoproteinssubstituted by those of subgroup B and RSV without the M2-2 gene areattenuated in African green monkeys” Virology 283:59-68) or theinsertion of the 9320 G gene in the A2 strain (Jin et al. (1998)Virology 251:206-214), subgroup B RSV vaccine development currently lagssignificantly behind subgroup A vaccine development. Availability of thesubgroup B RSV rescue system described herein permits manipulation ofthe subgroup B RSV genome, e.g., for vaccine development. For example,RSV B9320 can optionally be attenuated by methods used to attenuatesubgroup A strains. The methods that have been used to attenuate RSVsubgroup A RSV include, e.g., mutagenesis of the viral internal proteins(Lu et al. (2002) “Identification of temperature-sensitive mutations inthe phosphoprotein of respiratory syncytial virus that are likelyinvolved in its interaction with the nucleoprotein” J Virol76:2871-2880; Lu et al. (2002) “The major phosphorylation sites of therespiratory syncytial virus phosphoprotein are dispensable for virusreplication in vitro” J Virol 76:10776-10784; Tang et al. (2002)“Clustered charged-to-alanine mutagenesis of human respiratory syncytialvirus L polymerase generated temperature-sensitive viruses” Virology302(207-216); and Tang et al. (2001) “Requirement of cysteines andlength of the human respiratory syncytial virus M2-1 protein for proteinfunction and virus viability” J Virol 75:11328-11335), deletion of theaccessory genes (Bermingham and Collins (1999) Proc. Natl. Acad. Sci.USA 96:11259-11264; Jin et al. (2000) J. Virol. 74:74-82; Jin et al.(2000) Virology 273:210-218; Bukreyev et al. (1997) J Virol71:8973-8982; Teng and Collins (1999) J Virol 73:466-473); andintroduction of attenuating mutations from different strains tofine-tune the level of attenuation of a vaccine strain (Firestone et al.(1996) “Nucleotide sequence analysis of the respiratory syncytial virussubgroup A cold-passaged (cp) temperature sensitive (ts) cpts-248/404live attenuated virus vaccine candidate” Virology 225:419-422 andWhitehead et al. (1999) “Addition of a missense mutation present in theL gene of respiratory syncytial virus (RSV) cpts530/1030 to RSV vaccinecandidate cpts248/404 increases its attenuation and temperaturesensitivity” J Virol 73:871-877). As one example, from the gene deletionapproach, it appears that rA2ΔM2-2 exhibits some desired features forfurther clinical evaluation (Cheng et al. (2001) Virology 283:59-68; Jinet al. (2003) “Evaluation of recombinant respiratory syncytial virusgene deletion mutants in African green monkeys for their potential aslive attenuated vaccine candidates” Vaccine 21:3647-3652; and Teng etal. (2000) J Virol 74:9317-9321); 9320 with a deletion of the M2-2 geneis thus an attenuated 9320 vaccine candidate. Similarly, forcing use ofthe second or third start codon of the A2 M2-2 mRNA (instead of thefirst or all three start codons) results in a decrease in M2-2 activity(U.S. patent application Ser. No. 10/672,302 (attorney docket number26-000320US), filed on Sep. 26, 2003, Jin et al. entitled “Functionalmutations in respiratory syncytial virus”). 9320 with a mutation (e.g.,substitution or deletion) in the first, second, and/or third start codonof M2-2 is thus an attenuated 9320 vaccine candidate.

[0267] As noted, recombinant 9320 viruses with complete or partialdeletions of the G gene, e.g., as described herein, are attenuated 9320vaccine candidates.

[0268] While the foregoing invention has been described in some detailfor purposes of clarity and understanding, it will be clear to oneskilled in the art from a reading of this disclosure that variouschanges in form and detail can be made without departing from the truescope of the invention. For example, all the compositions and techniquesdescribed above can be used in various combinations. All publications,patents, patent applications, and/or other documents cited in thisapplication are incorporated by reference in their entirety for allpurposes to the same extent as if each individual publication, patent,patent application, and/or other document were individually indicated tobe incorporated by reference for all purposes.

1 54 1 15225 DNA respiratory syncytial virus B 9320 1 acgcgaaaaaatgcgtacta caaacttgca cattcgaaaa aaatggggca aataagaatt 60 tgataagtgttatttaagtc taaccttttc aatcagaaat ggggtgcaat tcattgagca 120 tgataaaggttagattacaa aatttatttg acaatgacga agtagcattg ttaaaaataa 180 catgttatactgacaaatta attcttctga ccaatgcatt agccaaagca gcaatacata 240 caattaaattaaacggcata gtttttatac atgttataac aagcagtgaa gtgtgccctg 300 ataacaatattgtagtgaaa tctaacttta caacaatgcc aatattacaa aacggaggat 360 acatatgggaattgattgag ttgacacact gctctcaatt aaatggtcta atggatgata 420 attgtgaaatcaaattttct aaaagactaa gtgactcagt aatgactgat tatatgaatc 480 aaatatctgatttacttggg cttgatctca attcatgaat tgtgtttagt ctaattcaat 540 agacatgtgtttattaccat tttagttaat ataaaaactc atcaaagaga aatggggcaa 600 ataaactcacctaatcagtc aaatcatgag cactacaaat aacaacacta ctatgcaaag 660 attgatgatcacagacatga gacccctgtc gatggaatca ataataacat ctctcaccaa 720 agaaatcataacacacaaat tcatatactt gataaacaat gaatgtattg taagaaaact 780 tgatgaaagacaagctacat tcacattcct agtcaattat gagatgaagc tactacacaa 840 agtagggagtaccaaatata agaaatacac tgaatataat acaaaatatg gcactttccc 900 tatgcctatatttatcaatc atggcgggtt tctagaatgt attggcatta agcctacaaa 960 acacactcctataatataca aatatgacct caacccgtaa attccaacaa aaaactaacc 1020 catccaaactaagctattcc ttaaataaca gtgctcaaca gttaagaagg ggctaatcca 1080 ttttagtaattaaaaataaa ggtaaagcca ataacataaa ttggggcaaa tacaaagatg 1140 gctcttagcaaagtcaagtt aaatgataca ttaaataagg atcagctgct gtcatctagc 1200 aaatacactattcaacgtag tacaggagat aatattgaca ctcccaatta tgatgtgcaa 1260 aaacacttaaacaaactatg tggtatgcta ttaatcactg aagatgcaaa tcataaattc 1320 acaggattaataggtatgtt atatgctatg tccaggttag gaagggaaga cactataaag 1380 atacttaaagatgctggata tcatgttaaa gctaatggag tagatataac aacatatcgt 1440 caagatataaatggaaagga aatgaaattc gaagtattaa cattatcaag cttgacatca 1500 gaaatacaagtcaatattga gatagaatct agaaagtcct acaaaaaaat gctaaaagag 1560 atgggagaagtggctccaga atataggcat gattctccag actgtgggat gataatactg 1620 tgtatagctgcacttgtaat aaccaaatta gcagcaggag atagatcagg tcttacagca 1680 gtaattaggagggcaaacaa tgtcttaaaa aacgaaataa aacgctacaa gggcctcata 1740 ccaaaggatatagctaacag tttttatgaa gtgtttgaaa aacaccctca tcttatagat 1800 gtttttgtgcactttggcat tgcacaatca tccacaagag ggggtagtag agttgaagga 1860 atctttgcaggattatttat gaatgcctat ggttcagggc aagtaatgct aagatgggga 1920 gttttagccaaatctgtaaa aaatatcatg ctaggacatg ctagtgtcca ggcagaaatg 1980 gagcaagttgtggaagtcta tgagtatgca cagaagttgg gaggagaagc tggattctac 2040 catatattgaacaatccaaa agcatcattg ctgtcattaa ctcaatttcc taacttctca 2100 agtgtggtcctaggcaatgc agcaggtcta ggcataatgg gagagtatag aggtacacca 2160 agaaaccaggatctttatga tgcagccaaa gcatatgcag agcaactcaa agaaaatgga 2220 gtaataaactacagtgtatt agacttaaca gcagaagaat tggaggccat aaagcatcaa 2280 ctcaaccccaaagaagatga tgtagagctc taagttaaca aaaaatacgg ggcaaataag 2340 tcaacatggagaagtttgca cctgaatttc atggagaaga tgcaaataac aaagctacca 2400 aattcctagaatcaataaag ggcaagttcg catcatccaa agatcctaag aagaaagata 2460 gcataatatctgttaactca atagatatag aagtaactaa agagagcccg ataacatctg 2520 gcaccaacatcaacaatcca acaagtgaag ctgacagtac cccagaagcc aaaaccaact 2580 acccaagaaaacccctagta agcttcaaag aagatctcac cccaagtgac aacccctttt 2640 ctaagttgtacaaagaaaca atagaaacat ttgataacaa tgaagaagaa tctagctact 2700 catatgaagaaataaatgat caaacaaatg acaacattac agcaagacta gatagaattg 2760 atgaaaaattaagtgaaata ttaggaatgc tccatacatt agtagttgca agtgcaggac 2820 ccacttcagctcgcgatgga ataagagatg ctatggttgg tctaagagaa gaaatgatag 2880 aaaaaataagagcggaagca ttaatgacca atgataggtt agaggctatg gcaagactta 2940 ggaatgaggaaagcgaaaaa atggcaaaag acacctcaga tgaagtgtct ctcaatccaa 3000 cttccaaaaaattgagtgac ttgctggaag acaacgatag tgacaatgat ctatcacttg 3060 atgatttttgatcagtgatc aactcactca gcaatcaaca acatcaataa gacagacatc 3120 aatccattgaatcaactgcc agaccgaaca aacaaacgtt catcagcaga accaccaacc 3180 aatcaatcaaccaattgatc aatcagcaac ctaacaaaat taacaatata gtaacaaaaa 3240 aagaacaagatggggcaaat atggaaacat acgtgaacaa gcttcacgaa ggctccacat 3300 acacagcagctgttcagtac aatgttctag aaaaagatga tgatcctgca tcactaacaa 3360 tatgggtgcctatgttccag tcatctgtgc cagcagactt gctcataaaa gaacttgcaa 3420 gcatcaacatactagtgaag cagatctcta cgcccaaagg accttcacta cgagtcacga 3480 tcaactcaagaagcgctgtg ctggcacaaa tgcccagtaa ttttatcata agtgcaaatg 3540 tatcattagatgaaagaagc aaattagcat atgatgtaac tacaccttgt gaaatcaaag 3600 catgcagtctaacatgctta aaagtaaaaa gtatgctaac tacagtcaaa gatcttacca 3660 tgaaaacattcaaccccact catgagatta ttgctctatg tgaatttgaa aatattatga 3720 catcaaaaagagtaataata ccaacctatc taagatcaat tagtgtcaaa aacaaggacc 3780 tgaactcactagaaaatata gcaaccaccg aattcaaaaa tgctatcacc aatgcgaaaa 3840 ttattccctatgcaggatta gtattagtta tcacagttac tgacaataaa ggagcattca 3900 aatatatcaagccacagagt caatttatag tagatcttgg agcctaccta gaaaaagaga 3960 gcatatattatgtgactaca aattggaagc atacagctac acgtttttca atcaaaccac 4020 tagaggattaaacttaatta tcaacgctaa atgacaggtc cacatatatc ctcaaactac 4080 acactatatccaaacatcat gaacatctac actacacact tcatcacaca aaccaatccc 4140 acttaaaatccaaaatcact tccagccact atctgctaga cctagagtgc gaataggtaa 4200 ataaaaccaaaatatggggt aaatagacat tagttagagt tcaatcaatc tcaacaacca 4260 tttatactgctaattcaata catatactat aaatttcaaa atgggaaata catccatcac 4320 aatagaattcactagcaaat tttggcctta ttttacacta atacatatga tcttaactct 4380 aatctctttactaattataa tcactattat gattgcaata ctaaataagc taagtgaaca 4440 taaaacattctgtaacaaaa ctcttgaact aggacagatg tatcaaatca acacatagtg 4500 ttctaccatcatgctgtgtc aaattataat cctgtatatg taaacaaaca aatccaatct 4560 tctcacagagtcatggtggc gcaaagccac gccaactatc atggtagcat agagtagtta 4620 tttaaaaattaacataatga tgaattatta gtatgggatc aaaaacaaca ttggggcaaa 4680 tgcaaccatgtccaaacaca agagtcaacg cactgccagg actctagaaa agacctggga 4740 tactcttaatcatctaattg taatatcctc ttgtttatac agactaaacc taaaatctat 4800 agcacaaatagcactatcag ttttggcaat gataatctca acctctctca taattgcagc 4860 cataatattcatcatctctg ccaatcacaa agttacacta acaacggtta cagttcaaac 4920 aataaaaaaccacactgaaa aaaacatcac cacctacctt actcaagtct caccagaaag 4980 ggttagctcatccatacaac ctacaaccac atcaccaatc cacacaaatt cagctacaat 5040 atcaccaaatacaaaatcag aaacacacca tacaacaaca caagccaaaa gcagaatcac 5100 cacttcaacacagaccaaca agccaagcac aaaatcacgt tcaaaaaatc caccaaaaaa 5160 accaaaagatgattaccatt ttgaagtgtt caattttgtt ccctgtagta tatgtggcaa 5220 caatcaactttgcaaatcca tctgcaaaac aataccaagc aacaaaccaa agaaaaaacc 5280 aaccatcaaacccacaaaca aaccaaccgt caaaaccaca aacaaaagag acccaaaaac 5340 accagccaaaatgatgaaaa aagaaaccac caccaaccca acaaaaaaac caaccctcaa 5400 gaccacagaaggagacacca gcacctcaca atccactgtg ctcgacacaa ccacatcaaa 5460 acacacaatccaacagcaat ccctccactc aatcacctcc gaaaacacac ccaactccac 5520 acaaatacccacagcaaccg aggcctccac atcaaattct acttaaaaaa cctagtcaca 5580 tgcttagttattcaaaaact acatcttagc agagaaccgt gatctatcaa gcaagaatga 5640 aattaaacctggggcaaata accatggagt tgctgatcca caggtcaagt gcaatcttcc 5700 taactcttgctattaatgca ttgtacctca cctcaagtca gaacataact gaggagtttt 5760 accaatcgacatgtagtgca gttagcagag gttattttag tgctttaaga acaggttggt 5820 ataccagtgttataacaata gaattaagta atataaaaga aaccaaatgc aatggaactg 5880 acactaaagtaaaacttata aaacaagaat tagataagta taagaatgca gtaacagaat 5940 tacagctacttacgcaaaac acgccagctg ccaacaaccg ggccagaaga gaagcaccac 6000 agtacatgaactacacaatc aataccacta aaaacctaaa cgtatcaata agcaagaaga 6060 ggaaacgaagatttctggga ttcttgttag gtgtaggatc tgcaatagca agtggtatag 6120 ctgtatccaaagttctacac cttgaaggag aagtgaacaa aatcaaaaat gctttgttgt 6180 ctacaaacaaagctgtagtc agtctatcaa atggggtcag tgttttaacc agcaaagtgt 6240 tagatctcaagagttacata aataaccaat tattacccat agtaaatcaa cagagctgtc 6300 gcatctccaacattgaaaca gttatagaat tccagcagaa gaacagcaga ttgttggaaa 6360 tcaccagagaatttagtgtc aatgcaggtg taacaacacc tttaagcact tacatgttaa 6420 caaacagtgagttactatca ttgatcaatg atatgcctat aacaaatgat cagaaaaaat 6480 taatgtcaagcaatgtccag atagtaaggc aacaaagtta ttctatcatg tctataataa 6540 aggaagaagtccttgcatat gttgtacagc tacctatcta tggtgtaata gatacacctt 6600 gctggaaattacacacatca cctctatgca ccaccaacat caaagaagga tcaaatattt 6660 gtttaacaaggactgataga ggatggtatt gtgataatgc aggatcagta tccttcttcc 6720 cacaggctgacacttgcaaa gtgcagtcca atcgagtatt ttgtgacact atgaacagtt 6780 tgacattaccaagtgaagtc agcctttgta acactgacat attcaattcc aagtatgact 6840 gcaaaatcatgacttcaaaa acagacataa gcagctcagt aattacttct cttggagcta 6900 tagtgtcatgctatggtaaa actaaatgca ctgcatccaa taaaaatcgt gggattataa 6960 agacattttctaatggttgt gactatgtgt caaacaaagg agtagatact gtgtcagtgg 7020 gcaacactttatactatgta aacaagctgg aaggcaaaaa cctttatgta aaaggggaac 7080 ctataataaattactatgat cctctagtgt ttccttctga tgagtttgat gcatcaatat 7140 ctcaagtcaatgaaaaaatc aatcaaagtt tagcttttat acgtagatct gatgaattac 7200 tacataatgtaaatactggc aaatctacta caaatattat gataaccaca atcattatag 7260 taatcattgtagtattgtta tcattaatag ctattggttt actgttgtat tgcaaagcta 7320 aaaacacaccagttacacta agcaaagacc aactaagtgg aatcaacaat attgcattca 7380 gcaaatagacaaaaaaccac ttgatcatgt ttcaacaaca atctgctgac caccaatccc 7440 aaatcaacttaacaacaaat atttcaacat catagcacag gctgaatcat ttcctcacat 7500 catgctacctacacaactaa gctagatcct taactcatag ttacataaaa acctcaagta 7560 tcacaatcaaacactaaatc gacacatcat tcacaaaatt aacaactggg gcaaatatgt 7620 cgcgaagaaatccttgtaaa tttgagatta gaggtcattg cttgaatggt agaagatgtc 7680 actacagtcataattatttt gaatggcctc ctcatgcatt actagtgagg caaaacttca 7740 tgttaaacaagatacttaag tcaatggaca aaagcataga cactttgtcg gaaataagtg 7800 gagctgctgaactggataga acagaagaat atgctcttgg tatagttgga gtgctagaga 7860 gttacataggatctataaac aacataacaa aacaatcagc atgtgttgct atgagtaaac 7920 ttcttattgagatcaacagt gatgacatta aaaaactgag agataatgaa gaacccaatt 7980 cacctaagataagagtgtac aatactgtta tatcatacat tgagagcaat agaaaaaaca 8040 acaagcaaaccatccatctg ctcaaaagac taccagcaga tgtgctgaag aagacaataa 8100 agaacacattagatatccac aaaagcataa ccataagcaa cccaaaagag tcaaccgtga 8160 atgatcaaaatgaccaaacc aaaaataatg atattaccgg ataaatatcc ttgtagtata 8220 tcatccatactgatttcaag tgaaagcatg gttgccacat tcaatcacaa aaacatatta 8280 caatttaaccataaccattt ggataaccac cagtgtttat taaatcatat atttgatgaa 8340 attcattggacacctaaaaa cttattagat accactcaac aatttctcca acatcttaac 8400 atccctgaagatatatatac agtatatata ttagtgtcat aatgcttgac cataacgatc 8460 ttatatcatccaaccataaa actatcataa taaggttatg ggacaaaatg gatcccatta 8520 ttaatggaaactctgctaat gtgtatctaa ctgatagtta tctaaaaggt gttatctctt 8580 tttcagaatgtaatgcttta gggagttacc tttttaacgg cccttatctt aaaaatgatt 8640 acactaacttaattagtaga caaagcccac tactagagca tatgaatcta aaaaaactaa 8700 ctataacacagtcattaata tctagatatc ataaaggtga actgaaatta gaagaaccaa 8760 cttatttccagtcattactt atgacatata aaagtatgtc ctcgtctgaa caaattgcta 8820 caactaacttacttaaaaaa ataatacgaa gagctataga aataagtgat gtaaaggtgt 8880 acgccatcttgaataaacta ggactaaagg aaaaggacag agttaagccc aacaataatt 8940 caggtgatgaaaactcagtt cttacaacca taattaaaga tgatatactt tcggctgtgg 9000 aaaacaatcaatcatataca aattcagaca aaaatcactc agtgaaccaa aatatcacta 9060 tcaaaacaacactcttgaaa aaattgatgt gttcaatgca acatcctcca tcatggttaa 9120 tacactggttcaatttatat acaaaattaa ataacatatt aacacaatat cgatcaaatg 9180 aggtaaaaagtcatgggttt atattaatag ataatcaaac tttaagtggt tttcagttta 9240 ttttaaatcaatatggttgt attgtttatc ataaaggact taaaaaaatc acaactacta 9300 cttacaatcaatttttgaca tggaaagaca tcagccttag cagattaaat gtttgcttaa 9360 ttacttggataagtaattgt ttaaatacat taaataaaag cttagggctg agatgtggat 9420 tcaataatgttgtgttatca caattatttc tttatggaga ttgtatactg aaattatttc 9480 ataatgaaggcttctacata ataaaagaag tagagggatt tattatgtct ttaattctaa 9540 acataacagaagaagatcaa tttaggacac gattttataa cagcatgcta aataacatca 9600 cagatgcagctattaaggct caaaaaaacc tactatcaag agtatgtcac actttattgg 9660 acaagacagtgtctgataat atcataaatg gtaaatggat aatcctatta agtaaatttc 9720 ttaaattgattaagcttgca ggtgataata atctcaataa cttgagtgag ctatattttc 9780 tcttcagaatctttggacat ccaatggtcg atgaaagaca agcaatggat gctgtaagaa 9840 ttaactgtaatgaaactaag ttctacttat taagtagtct aagtacgtta agaggtgctt 9900 tcatttatagaatcataaaa gggtttgtaa atacctacaa cagatggccc actttaagga 9960 atgctattgttctacctcta agatggttga actattataa acttaatact tatccatctc 10020 tacttgaaatcacagaaaat gatttgatta ttttatcagg attgaggttc tatcgtgagt 10080 ttcatctgcctaaaaaagtg gatcttgaaa tgataataaa tgacaaagcc atttcacctc 10140 caaaagatctaatatggact agttttccca gaaattacat gccatcacat atacaaaatt 10200 atatagaacatgaaaagttg aagttctctg aaagcgacag atcaagaaga gtactagagt 10260 attacttgagagataataaa ttcaatgaat gcgatctata caattgtgtg gtcaatcaaa 10320 gctatctcaacaactctaac cacgtggtat cactaactgg taaagaaaga gagctcagtg 10380 taggtagaatgtttgctatg caaccaggta tgtttaggca aattcaaatc ttagcagaga 10440 aaatgatagccgaaaatatt ttacaattct tccctgagag tttgacaaga tatggtgatc 10500 tagagcttcaaaagatatta gaattaaaag caggaataag caacaaatca aatcgttata 10560 atgataactacaacaattat atcagtaaat gttctatcat tacagacctt agcaaattca 10620 atcaagcatttagatatgaa acatcatgta tctgcagtga tgtattagat gaactgcatg 10680 gagtacaatcactgttctct tggttgcatt taacaatacc tcttgtcaca ataatatgta 10740 catatagacatgcacctcct ttcataaagg atcatgttgt taatctgaat gaagttgatg 10800 aacaaagtggattatacaga tatcatatgg gtggtattga gggctggtgt caaaaactgt 10860 ggaccattgaagctatatca ttattagatc taatatccct caaagggaaa ttctctatca 10920 cagctctaataaatggtgat aatcagtcaa ttgatataag taaaccagtt agacttatag 10980 agggtcagacccatgctcaa gcagattatt tgttagcatt aaatagcctt aaattgctat 11040 ataaagagtatgcaggcata ggccataagc tcaagggaac agaaacctat atatcccgag 11100 atatgcaattcatgagcaaa acaatccagc acaatggagt gtactatcca gccagtatca 11160 aaaaagtcctgagagtaggt ccatggataa atacaatact tgatgatttt aaagttagtt 11220 tagaatctataggcagctta acacaggagt tagaatacag aggagaaagc ttattatgca 11280 gtttaatatttagaaacatt tggttataca atcaaattgc tttgcaactc cgaaatcatg 11340 cattatgtcacaataagcta tatttagata tattgaaagt attaaaacac ttaaaaactt 11400 tttttaatcttgatagtatc gatatggcat tatcattgta tatgaatttg cctatgctgt 11460 ttggtggtggtgatcctaat ttgttatatc gaagctttta tagaagaact ccagacttcc 11520 ttacagaagctatagtacat tcagtgtttg tgttgagcta ttatactggt cacgatttac 11580 aagataagctccaggatctt ccagatgata gactgaacaa attcttgaca tgtatcatca 11640 catttgataaaaatcccaat gccgagtttg taacattaat gagggatcca caggctttag 11700 ggtctgaaaggcaagctaaa attactagtg agattaatag attagcagta acggaagtct 11760 taagtatagctccaaacaaa atattttcta aaagtgcaca acattatact accactgaga 11820 ttgatctaaatgatattatg caaaatatag aaccaactta ccctcatgga ttaagagttg 11880 tttatgaaagtttacctttt tataaagcag aaaaaatagt taatcttata tcaggaacaa 11940 aatccataactaatatactt gaaaaaacat cagcaataga tacaactgat attaataggg 12000 ctactgatatgatgaggaaa aatataactt tacttataag gatacttcca ctagattgta 12060 acaaagacaaaagagagtta ttaagtttag aaaatcttag tataactgaa ttaagcaagt 12120 atgtaagagaaagatcttgg tcgttatcca atatagtagg agtaacatcg ccaagtatta 12180 tgttcacaatggacattaaa tatacaacta gcactatagc cagtggtata attatagaaa 12240 aatataatgttaatagttta actcgtggtg aaagaggacc tactaagcca tgggtaggtt 12300 catctacgcaggagaaaaaa acaatgccag tgtataatag acaagtttta accaaaaagc 12360 aaagagaccaaatagattta ttagcaaaat tagactgggt atatgcatcc atagacaaca 12420 aagatgaattcatggaagaa ctgagtactg gaacacttgg attgtcatat gaaaaagcca 12480 aaaaattgtttccacaatat ctaagtgtca attatttaca ccgcttaaca gtcagtagta 12540 ggccatgtgaattccctgca tcaataccag cttatagaac aacaaattat catttcgata 12600 ctagtcctatcaatcatgta ttaacagaaa agtatggaga tgaagatatc gacatagtgt 12660 ttcaaaattgcataagtttt ggtcttagcc taatgtcggt tgtggaacaa ttcacaaaca 12720 tatgtcctaatagaattatt ctcataccga agctgaatga gatacatttg atgaaacctc 12780 ctatatttacaggagatgtt gatatcatca aattgaagca agtgatacaa aaacagcaca 12840 tgttcctaccagataaaata agtttaaccc aatatgtaga attattccta agtaacaaag 12900 cacttaaatctggatctcac atcaactcta atttaatatt agtacataaa atgtctgatt 12960 attttcataatgattatatt ttaagtacta atttagctgg acattggatt ctgattattc 13020 aacttatgaaagattcaaaa ggtatttttg aaaaagattg gggagagggg tatataactg 13080 atcatatgttcattaatttg aatgttttct ttaatgctta taagacttat ttgctatgtt 13140 ttcataaaggttatggtaaa gcaaaattag aatgtgatat gaacacttca gatcttcttt 13200 gtgttttggagttaatagac agtagctact ggaaatctat gtctaaagtt ttcctagaac 13260 agaaagtcataaaatacata gtcaatcaag acacaagttt gcatagaata aaaggttgtc 13320 atagttttaagttgtggttt ttaaaacgcc ttaataatgc taaatttacc gtatgccctt 13380 gggttgttaacatagattat cacccaacac acatgaaagc tatattatct tacatagatt 13440 tagttagaatggggttaata aatgtagata aattaaccat taaaaataaa aacaaattca 13500 atgatgaattttacacatca aatctctttt atattagtta taacttttca gacaacactc 13560 atttgctaacaaaacaaata agaattgcta attcagaatt agaaaataat tataacaaac 13620 tatatcacccaaccccagaa actttagaaa atatgtcatt aattcctgtt aaaagtaaca 13680 atagtaacaaacctaaatct tgtataagtg gaaataccga atctatgatg acgtcaacat 13740 tctccaataaaatgcatatt aaatcttcca ctgttaccac aagattaaac tatagcaaac 13800 aagacttgtacaatttattt ccaattgttg tgatagacag gattatagat cattcaggca 13860 atacagcaaaatccaaccaa ctttacacca ccacttcaca tcagacatct ttagtaagga 13920 atagtgcatcactttattgc atgcttcctt ggcatcatgt caatagattt aactttgtat 13980 ttagttccacaggatgcaag atcagtatag agtatatttt aaaagatctt aagattaagg 14040 accccagttgtatagcattc ataggtgaag gagctggtaa cttattatta cgtacggtag 14100 tagaacttcatccagacata agatacattt acagaagttt aaaagattgc aatgatcata 14160 gtttacctattgaatttcta aggttataca acgggcatat aaacatagat tatggtgaga 14220 atttaaccattcctgctaca gatgcaacta ataacattca ttggtcttat ttacatataa 14280 aatttgcagaacctattagc atctttgtct gcgatgctga attacctgtt acagccaatt 14340 ggagtaaaattataattgaa tggagtaagc atgtaagaaa gtgcaagtac tgttcctctg 14400 taaatagatgcattttaatt gcaaaatatc atgctcaaga tgatattgat ttcaaattag 14460 ataacattactatattaaaa acttatgtgt gcctaggtag caagttaaaa ggatctgaag 14520 tttacttagtccttacaata ggcccttcaa atatacttcc tgtttttaat gttgtgcaaa 14580 atgctaaattgattctttca agaactaaaa atttcattat gcctaaaaaa actgacaaag 14640 aatctatcgatgcaaatatt aaaagcttaa tacctttcct ttgttaccct ataacaaaaa 14700 aaggaattaagacttcattg tcaaaattga agagtgtagt taatggagat atattatcat 14760 attctatagctggacgtaat gaagtattca gcaacaagct tataaaccac aagcatatga 14820 atatcctaaaatggctagat catgttttaa actttagatc aactgaactt aattacaatc 14880 atttatatatgatagagtcc acatatcctt acttaagtga attgttaaat agtttaacaa 14940 ccaatgagctcaaaaagctg attaaaatta caggtagtgt actatacaac cttcccaatg 15000 aacagtaacttaaaatatca ttaacaagtt tggtcaaatt tagatgctaa cacatcatta 15060 tattatagttattaaaaaat atgcaaactt ttcaataatt tagcatattg attccaaaat 15120 tatctattttggtcttaagg ggttaaataa aaatctaaaa ctaacaatta tacatgtgca 15180 tttacaacacaacgagacat tagtttttga cacttttttt ctcgt 15225 2 139 PRT respiratorysyncytial virus B 9320 2 Met Gly Cys Asn Ser Leu Ser Met Ile Lys Val ArgLeu Gln Asn Leu 1 5 10 15 Phe Asp Asn Asp Glu Val Ala Leu Leu Lys IleThr Cys Tyr Thr Asp 20 25 30 Lys Leu Ile Leu Leu Thr Asn Ala Leu Ala LysAla Ala Ile His Thr 35 40 45 Ile Lys Leu Asn Gly Ile Val Phe Ile His ValIle Thr Ser Ser Glu 50 55 60 Val Cys Pro Asp Asn Asn Ile Val Val Lys SerAsn Phe Thr Thr Met 65 70 75 80 Pro Ile Leu Gln Asn Gly Gly Tyr Ile TrpGlu Leu Ile Glu Leu Thr 85 90 95 His Cys Ser Gln Leu Asn Gly Leu Met AspAsp Asn Cys Glu Ile Lys 100 105 110 Phe Ser Lys Arg Leu Ser Asp Ser ValMet Thr Asp Tyr Met Asn Gln 115 120 125 Ile Ser Asp Leu Leu Gly Leu AspLeu Asn Ser 130 135 3 124 PRT respiratory syncytial virus B 9320 3 MetSer Thr Thr Asn Asn Asn Thr Thr Met Gln Arg Leu Met Ile Thr 1 5 10 15Asp Met Arg Pro Leu Ser Met Glu Ser Ile Ile Thr Ser Leu Thr Lys 20 25 30Glu Ile Ile Thr His Lys Phe Ile Tyr Leu Ile Asn Asn Glu Cys Ile 35 40 45Val Arg Lys Leu Asp Glu Arg Gln Ala Thr Phe Thr Phe Leu Val Asn 50 55 60Tyr Glu Met Lys Leu Leu His Lys Val Gly Ser Thr Lys Tyr Lys Lys 65 70 7580 Tyr Thr Glu Tyr Asn Thr Lys Tyr Gly Thr Phe Pro Met Pro Ile Phe 85 9095 Ile Asn His Gly Gly Phe Leu Glu Cys Ile Gly Ile Lys Pro Thr Lys 100105 110 His Thr Pro Ile Ile Tyr Lys Tyr Asp Leu Asn Pro 115 120 4 391PRT respiratory syncytial virus B 9320 4 Met Ala Leu Ser Lys Val Lys LeuAsn Asp Thr Leu Asn Lys Asp Gln 1 5 10 15 Leu Leu Ser Ser Ser Lys TyrThr Ile Gln Arg Ser Thr Gly Asp Asn 20 25 30 Ile Asp Thr Pro Asn Tyr AspVal Gln Lys His Leu Asn Lys Leu Cys 35 40 45 Gly Met Leu Leu Ile Thr GluAsp Ala Asn His Lys Phe Thr Gly Leu 50 55 60 Ile Gly Met Leu Tyr Ala MetSer Arg Leu Gly Arg Glu Asp Thr Ile 65 70 75 80 Lys Ile Leu Lys Asp AlaGly Tyr His Val Lys Ala Asn Gly Val Asp 85 90 95 Ile Thr Thr Tyr Arg GlnAsp Ile Asn Gly Lys Glu Met Lys Phe Glu 100 105 110 Val Leu Thr Leu SerSer Leu Thr Ser Glu Ile Gln Val Asn Ile Glu 115 120 125 Ile Glu Ser ArgLys Ser Tyr Lys Lys Met Leu Lys Glu Met Gly Glu 130 135 140 Val Ala ProGlu Tyr Arg His Asp Ser Pro Asp Cys Gly Met Ile Ile 145 150 155 160 LeuCys Ile Ala Ala Leu Val Ile Thr Lys Leu Ala Ala Gly Asp Arg 165 170 175Ser Gly Leu Thr Ala Val Ile Arg Arg Ala Asn Asn Val Leu Lys Asn 180 185190 Glu Ile Lys Arg Tyr Lys Gly Leu Ile Pro Lys Asp Ile Ala Asn Ser 195200 205 Phe Tyr Glu Val Phe Glu Lys His Pro His Leu Ile Asp Val Phe Val210 215 220 His Phe Gly Ile Ala Gln Ser Ser Thr Arg Gly Gly Ser Arg ValGlu 225 230 235 240 Gly Ile Phe Ala Gly Leu Phe Met Asn Ala Tyr Gly SerGly Gln Val 245 250 255 Met Leu Arg Trp Gly Val Leu Ala Lys Ser Val LysAsn Ile Met Leu 260 265 270 Gly His Ala Ser Val Gln Ala Glu Met Glu GlnVal Val Glu Val Tyr 275 280 285 Glu Tyr Ala Gln Lys Leu Gly Gly Glu AlaGly Phe Tyr His Ile Leu 290 295 300 Asn Asn Pro Lys Ala Ser Leu Leu SerLeu Thr Gln Phe Pro Asn Phe 305 310 315 320 Ser Ser Val Val Leu Gly AsnAla Ala Gly Leu Gly Ile Met Gly Glu 325 330 335 Tyr Arg Gly Thr Pro ArgAsn Gln Asp Leu Tyr Asp Ala Ala Lys Ala 340 345 350 Tyr Ala Glu Gln LeuLys Glu Asn Gly Val Ile Asn Tyr Ser Val Leu 355 360 365 Asp Leu Thr AlaGlu Glu Leu Glu Ala Ile Lys His Gln Leu Asn Pro 370 375 380 Lys Glu AspAsp Val Glu Leu 385 390 5 241 PRT respiratory syncytial virus B 9320 5Met Glu Lys Phe Ala Pro Glu Phe His Gly Glu Asp Ala Asn Asn Lys 1 5 1015 Ala Thr Lys Phe Leu Glu Ser Ile Lys Gly Lys Phe Ala Ser Ser Lys 20 2530 Asp Pro Lys Lys Lys Asp Ser Ile Ile Ser Val Asn Ser Ile Asp Ile 35 4045 Glu Val Thr Lys Glu Ser Pro Ile Thr Ser Gly Thr Asn Ile Asn Asn 50 5560 Pro Thr Ser Glu Ala Asp Ser Thr Pro Glu Ala Lys Thr Asn Tyr Pro 65 7075 80 Arg Lys Pro Leu Val Ser Phe Lys Glu Asp Leu Thr Pro Ser Asp Asn 8590 95 Pro Phe Ser Lys Leu Tyr Lys Glu Thr Ile Glu Thr Phe Asp Asn Asn100 105 110 Glu Glu Glu Ser Ser Tyr Ser Tyr Glu Glu Ile Asn Asp Gln ThrAsn 115 120 125 Asp Asn Ile Thr Ala Arg Leu Asp Arg Ile Asp Glu Lys LeuSer Glu 130 135 140 Ile Leu Gly Met Leu His Thr Leu Val Val Ala Ser AlaGly Pro Thr 145 150 155 160 Ser Ala Arg Asp Gly Ile Arg Asp Ala Met ValGly Leu Arg Glu Glu 165 170 175 Met Ile Glu Lys Ile Arg Ala Glu Ala LeuMet Thr Asn Asp Arg Leu 180 185 190 Glu Ala Met Ala Arg Leu Arg Asn GluGlu Ser Glu Lys Met Ala Lys 195 200 205 Asp Thr Ser Asp Glu Val Ser LeuAsn Pro Thr Ser Lys Lys Leu Ser 210 215 220 Asp Leu Leu Glu Asp Asn AspSer Asp Asn Asp Leu Ser Leu Asp Asp 225 230 235 240 Phe 6 256 PRTrespiratory syncytial virus B 9320 6 Met Glu Thr Tyr Val Asn Lys Leu HisGlu Gly Ser Thr Tyr Thr Ala 1 5 10 15 Ala Val Gln Tyr Asn Val Leu GluLys Asp Asp Asp Pro Ala Ser Leu 20 25 30 Thr Ile Trp Val Pro Met Phe GlnSer Ser Val Pro Ala Asp Leu Leu 35 40 45 Ile Lys Glu Leu Ala Ser Ile AsnIle Leu Val Lys Gln Ile Ser Thr 50 55 60 Pro Lys Gly Pro Ser Leu Arg ValThr Ile Asn Ser Arg Ser Ala Val 65 70 75 80 Leu Ala Gln Met Pro Ser AsnPhe Ile Ile Ser Ala Asn Val Ser Leu 85 90 95 Asp Glu Arg Ser Lys Leu AlaTyr Asp Val Thr Thr Pro Cys Glu Ile 100 105 110 Lys Ala Cys Ser Leu ThrCys Leu Lys Val Lys Ser Met Leu Thr Thr 115 120 125 Val Lys Asp Leu ThrMet Lys Thr Phe Asn Pro Thr His Glu Ile Ile 130 135 140 Ala Leu Cys GluPhe Glu Asn Ile Met Thr Ser Lys Arg Val Ile Ile 145 150 155 160 Pro ThrTyr Leu Arg Ser Ile Ser Val Lys Asn Lys Asp Leu Asn Ser 165 170 175 LeuGlu Asn Ile Ala Thr Thr Glu Phe Lys Asn Ala Ile Thr Asn Ala 180 185 190Lys Ile Ile Pro Tyr Ala Gly Leu Val Leu Val Ile Thr Val Thr Asp 195 200205 Asn Lys Gly Ala Phe Lys Tyr Ile Lys Pro Gln Ser Gln Phe Ile Val 210215 220 Asp Leu Gly Ala Tyr Leu Glu Lys Glu Ser Ile Tyr Tyr Val Thr Thr225 230 235 240 Asn Trp Lys His Thr Ala Thr Arg Phe Ser Ile Lys Pro LeuGlu Asp 245 250 255 7 65 PRT respiratory syncytial virus B 9320 7 MetGly Asn Thr Ser Ile Thr Ile Glu Phe Thr Ser Lys Phe Trp Pro 1 5 10 15Tyr Phe Thr Leu Ile His Met Ile Leu Thr Leu Ile Ser Leu Leu Ile 20 25 30Ile Ile Thr Ile Met Ile Ala Ile Leu Asn Lys Leu Ser Glu His Lys 35 40 45Thr Phe Cys Asn Lys Thr Leu Glu Leu Gly Gln Met Tyr Gln Ile Asn 50 55 60Thr 65 8 574 PRT respiratory syncytial virus B 9320 8 Met Glu Leu LeuIle His Arg Ser Ser Ala Ile Phe Leu Thr Leu Ala 1 5 10 15 Ile Asn AlaLeu Tyr Leu Thr Ser Ser Gln Asn Ile Thr Glu Glu Phe 20 25 30 Tyr Gln SerThr Cys Ser Ala Val Ser Arg Gly Tyr Phe Ser Ala Leu 35 40 45 Arg Thr GlyTrp Tyr Thr Ser Val Ile Thr Ile Glu Leu Ser Asn Ile 50 55 60 Lys Glu ThrLys Cys Asn Gly Thr Asp Thr Lys Val Lys Leu Ile Lys 65 70 75 80 Gln GluLeu Asp Lys Tyr Lys Asn Ala Val Thr Glu Leu Gln Leu Leu 85 90 95 Thr GlnAsn Thr Pro Ala Ala Asn Asn Arg Ala Arg Arg Glu Ala Pro 100 105 110 GlnTyr Met Asn Tyr Thr Ile Asn Thr Thr Lys Asn Leu Asn Val Ser 115 120 125Ile Ser Lys Lys Arg Lys Arg Arg Phe Leu Gly Phe Leu Leu Gly Val 130 135140 Gly Ser Ala Ile Ala Ser Gly Ile Ala Val Ser Lys Val Leu His Leu 145150 155 160 Glu Gly Glu Val Asn Lys Ile Lys Asn Ala Leu Leu Ser Thr AsnLys 165 170 175 Ala Val Val Ser Leu Ser Asn Gly Val Ser Val Leu Thr SerLys Val 180 185 190 Leu Asp Leu Lys Ser Tyr Ile Asn Asn Gln Leu Leu ProIle Val Asn 195 200 205 Gln Gln Ser Cys Arg Ile Ser Asn Ile Glu Thr ValIle Glu Phe Gln 210 215 220 Gln Lys Asn Ser Arg Leu Leu Glu Ile Thr ArgGlu Phe Ser Val Asn 225 230 235 240 Ala Gly Val Thr Thr Pro Leu Ser ThrTyr Met Leu Thr Asn Ser Glu 245 250 255 Leu Leu Ser Leu Ile Asn Asp MetPro Ile Thr Asn Asp Gln Lys Lys 260 265 270 Leu Met Ser Ser Asn Val GlnIle Val Arg Gln Gln Ser Tyr Ser Ile 275 280 285 Met Ser Ile Ile Lys GluGlu Val Leu Ala Tyr Val Val Gln Leu Pro 290 295 300 Ile Tyr Gly Val IleAsp Thr Pro Cys Trp Lys Leu His Thr Ser Pro 305 310 315 320 Leu Cys ThrThr Asn Ile Lys Glu Gly Ser Asn Ile Cys Leu Thr Arg 325 330 335 Thr AspArg Gly Trp Tyr Cys Asp Asn Ala Gly Ser Val Ser Phe Phe 340 345 350 ProGln Ala Asp Thr Cys Lys Val Gln Ser Asn Arg Val Phe Cys Asp 355 360 365Thr Met Asn Ser Leu Thr Leu Pro Ser Glu Val Ser Leu Cys Asn Thr 370 375380 Asp Ile Phe Asn Ser Lys Tyr Asp Cys Lys Ile Met Thr Ser Lys Thr 385390 395 400 Asp Ile Ser Ser Ser Val Ile Thr Ser Leu Gly Ala Ile Val SerCys 405 410 415 Tyr Gly Lys Thr Lys Cys Thr Ala Ser Asn Lys Asn Arg GlyIle Ile 420 425 430 Lys Thr Phe Ser Asn Gly Cys Asp Tyr Val Ser Asn LysGly Val Asp 435 440 445 Thr Val Ser Val Gly Asn Thr Leu Tyr Tyr Val AsnLys Leu Glu Gly 450 455 460 Lys Asn Leu Tyr Val Lys Gly Glu Pro Ile IleAsn Tyr Tyr Asp Pro 465 470 475 480 Leu Val Phe Pro Ser Asp Glu Phe AspAla Ser Ile Ser Gln Val Asn 485 490 495 Glu Lys Ile Asn Gln Ser Leu AlaPhe Ile Arg Arg Ser Asp Glu Leu 500 505 510 Leu His Asn Val Asn Thr GlyLys Ser Thr Thr Asn Ile Met Ile Thr 515 520 525 Thr Ile Ile Ile Val IleIle Val Val Leu Leu Ser Leu Ile Ala Ile 530 535 540 Gly Leu Leu Leu TyrCys Lys Ala Lys Asn Thr Pro Val Thr Leu Ser 545 550 555 560 Lys Asp GlnLeu Ser Gly Ile Asn Asn Ile Ala Phe Ser Lys 565 570 9 195 PRTrespiratory syncytial virus B 9320 9 Met Ser Arg Arg Asn Pro Cys Lys PheGlu Ile Arg Gly His Cys Leu 1 5 10 15 Asn Gly Arg Arg Cys His Tyr SerHis Asn Tyr Phe Glu Trp Pro Pro 20 25 30 His Ala Leu Leu Val Arg Gln AsnPhe Met Leu Asn Lys Ile Leu Lys 35 40 45 Ser Met Asp Lys Ser Ile Asp ThrLeu Ser Glu Ile Ser Gly Ala Ala 50 55 60 Glu Leu Asp Arg Thr Glu Glu TyrAla Leu Gly Ile Val Gly Val Leu 65 70 75 80 Glu Ser Tyr Ile Gly Ser IleAsn Asn Ile Thr Lys Gln Ser Ala Cys 85 90 95 Val Ala Met Ser Lys Leu LeuIle Glu Ile Asn Ser Asp Asp Ile Lys 100 105 110 Lys Leu Arg Asp Asn GluGlu Pro Asn Ser Pro Lys Ile Arg Val Tyr 115 120 125 Asn Thr Val Ile SerTyr Ile Glu Ser Asn Arg Lys Asn Asn Lys Gln 130 135 140 Thr Ile His LeuLeu Lys Arg Leu Pro Ala Asp Val Leu Lys Lys Thr 145 150 155 160 Ile LysAsn Thr Leu Asp Ile His Lys Ser Ile Thr Ile Ser Asn Pro 165 170 175 LysGlu Ser Thr Val Asn Asp Gln Asn Asp Gln Thr Lys Asn Asn Asp 180 185 190Ile Thr Gly 195 10 93 PRT respiratory syncytial virus B 9320 10 Met IleLys Met Thr Lys Pro Lys Ile Met Ile Leu Pro Asp Lys Tyr 1 5 10 15 ProCys Ser Ile Ser Ser Ile Leu Ile Ser Ser Glu Ser Met Val Ala 20 25 30 ThrPhe Asn His Lys Asn Ile Leu Gln Phe Asn His Asn His Leu Asp 35 40 45 AsnHis Gln Cys Leu Leu Asn His Ile Phe Asp Glu Ile His Trp Thr 50 55 60 ProLys Asn Leu Leu Asp Thr Thr Gln Gln Phe Leu Gln His Leu Asn 65 70 75 80Ile Pro Glu Asp Ile Tyr Thr Val Tyr Ile Leu Val Ser 85 90 11 2166 PRTrespiratory syncytial virus B 9320 11 Met Asp Pro Ile Ile Asn Gly AsnSer Ala Asn Val Tyr Leu Thr Asp 1 5 10 15 Ser Tyr Leu Lys Gly Val IleSer Phe Ser Glu Cys Asn Ala Leu Gly 20 25 30 Ser Tyr Leu Phe Asn Gly ProTyr Leu Lys Asn Asp Tyr Thr Asn Leu 35 40 45 Ile Ser Arg Gln Ser Pro LeuLeu Glu His Met Asn Leu Lys Lys Leu 50 55 60 Thr Ile Thr Gln Ser Leu IleSer Arg Tyr His Lys Gly Glu Leu Lys 65 70 75 80 Leu Glu Glu Pro Thr TyrPhe Gln Ser Leu Leu Met Thr Tyr Lys Ser 85 90 95 Met Ser Ser Ser Glu GlnIle Ala Thr Thr Asn Leu Leu Lys Lys Ile 100 105 110 Ile Arg Arg Ala IleGlu Ile Ser Asp Val Lys Val Tyr Ala Ile Leu 115 120 125 Asn Lys Leu GlyLeu Lys Glu Lys Asp Arg Val Lys Pro Asn Asn Asn 130 135 140 Ser Gly AspGlu Asn Ser Val Leu Thr Thr Ile Ile Lys Asp Asp Ile 145 150 155 160 LeuSer Ala Val Glu Asn Asn Gln Ser Tyr Thr Asn Ser Asp Lys Asn 165 170 175His Ser Val Asn Gln Asn Ile Thr Ile Lys Thr Thr Leu Leu Lys Lys 180 185190 Leu Met Cys Ser Met Gln His Pro Pro Ser Trp Leu Ile His Trp Phe 195200 205 Asn Leu Tyr Thr Lys Leu Asn Asn Ile Leu Thr Gln Tyr Arg Ser Asn210 215 220 Glu Val Lys Ser His Gly Phe Ile Leu Ile Asp Asn Gln Thr LeuSer 225 230 235 240 Gly Phe Gln Phe Ile Leu Asn Gln Tyr Gly Cys Ile ValTyr His Lys 245 250 255 Gly Leu Lys Lys Ile Thr Thr Thr Thr Tyr Asn GlnPhe Leu Thr Trp 260 265 270 Lys Asp Ile Ser Leu Ser Arg Leu Asn Val CysLeu Ile Thr Trp Ile 275 280 285 Ser Asn Cys Leu Asn Thr Leu Asn Lys SerLeu Gly Leu Arg Cys Gly 290 295 300 Phe Asn Asn Val Val Leu Ser Gln LeuPhe Leu Tyr Gly Asp Cys Ile 305 310 315 320 Leu Lys Leu Phe His Asn GluGly Phe Tyr Ile Ile Lys Glu Val Glu 325 330 335 Gly Phe Ile Met Ser LeuIle Leu Asn Ile Thr Glu Glu Asp Gln Phe 340 345 350 Arg Thr Arg Phe TyrAsn Ser Met Leu Asn Asn Ile Thr Asp Ala Ala 355 360 365 Ile Lys Ala GlnLys Asn Leu Leu Ser Arg Val Cys His Thr Leu Leu 370 375 380 Asp Lys ThrVal Ser Asp Asn Ile Ile Asn Gly Lys Trp Ile Ile Leu 385 390 395 400 LeuSer Lys Phe Leu Lys Leu Ile Lys Leu Ala Gly Asp Asn Asn Leu 405 410 415Asn Asn Leu Ser Glu Leu Tyr Phe Leu Phe Arg Ile Phe Gly His Pro 420 425430 Met Val Asp Glu Arg Gln Ala Met Asp Ala Val Arg Ile Asn Cys Asn 435440 445 Glu Thr Lys Phe Tyr Leu Leu Ser Ser Leu Ser Thr Leu Arg Gly Ala450 455 460 Phe Ile Tyr Arg Ile Ile Lys Gly Phe Val Asn Thr Tyr Asn ArgTrp 465 470 475 480 Pro Thr Leu Arg Asn Ala Ile Val Leu Pro Leu Arg TrpLeu Asn Tyr 485 490 495 Tyr Lys Leu Asn Thr Tyr Pro Ser Leu Leu Glu IleThr Glu Asn Asp 500 505 510 Leu Ile Ile Leu Ser Gly Leu Arg Phe Tyr ArgGlu Phe His Leu Pro 515 520 525 Lys Lys Val Asp Leu Glu Met Ile Ile AsnAsp Lys Ala Ile Ser Pro 530 535 540 Pro Lys Asp Leu Ile Trp Thr Ser PhePro Arg Asn Tyr Met Pro Ser 545 550 555 560 His Ile Gln Asn Tyr Ile GluHis Glu Lys Leu Lys Phe Ser Glu Ser 565 570 575 Asp Arg Ser Arg Arg ValLeu Glu Tyr Tyr Leu Arg Asp Asn Lys Phe 580 585 590 Asn Glu Cys Asp LeuTyr Asn Cys Val Val Asn Gln Ser Tyr Leu Asn 595 600 605 Asn Ser Asn HisVal Val Ser Leu Thr Gly Lys Glu Arg Glu Leu Ser 610 615 620 Val Gly ArgMet Phe Ala Met Gln Pro Gly Met Phe Arg Gln Ile Gln 625 630 635 640 IleLeu Ala Glu Lys Met Ile Ala Glu Asn Ile Leu Gln Phe Phe Pro 645 650 655Glu Ser Leu Thr Arg Tyr Gly Asp Leu Glu Leu Gln Lys Ile Leu Glu 660 665670 Leu Lys Ala Gly Ile Ser Asn Lys Ser Asn Arg Tyr Asn Asp Asn Tyr 675680 685 Asn Asn Tyr Ile Ser Lys Cys Ser Ile Ile Thr Asp Leu Ser Lys Phe690 695 700 Asn Gln Ala Phe Arg Tyr Glu Thr Ser Cys Ile Cys Ser Asp ValLeu 705 710 715 720 Asp Glu Leu His Gly Val Gln Ser Leu Phe Ser Trp LeuHis Leu Thr 725 730 735 Ile Pro Leu Val Thr Ile Ile Cys Thr Tyr Arg HisAla Pro Pro Phe 740 745 750 Ile Lys Asp His Val Val Asn Leu Asn Glu ValAsp Glu Gln Ser Gly 755 760 765 Leu Tyr Arg Tyr His Met Gly Gly Ile GluGly Trp Cys Gln Lys Leu 770 775 780 Trp Thr Ile Glu Ala Ile Ser Leu LeuAsp Leu Ile Ser Leu Lys Gly 785 790 795 800 Lys Phe Ser Ile Thr Ala LeuIle Asn Gly Asp Asn Gln Ser Ile Asp 805 810 815 Ile Ser Lys Pro Val ArgLeu Ile Glu Gly Gln Thr His Ala Gln Ala 820 825 830 Asp Tyr Leu Leu AlaLeu Asn Ser Leu Lys Leu Leu Tyr Lys Glu Tyr 835 840 845 Ala Gly Ile GlyHis Lys Leu Lys Gly Thr Glu Thr Tyr Ile Ser Arg 850 855 860 Asp Met GlnPhe Met Ser Lys Thr Ile Gln His Asn Gly Val Tyr Tyr 865 870 875 880 ProAla Ser Ile Lys Lys Val Leu Arg Val Gly Pro Trp Ile Asn Thr 885 890 895Ile Leu Asp Asp Phe Lys Val Ser Leu Glu Ser Ile Gly Ser Leu Thr 900 905910 Gln Glu Leu Glu Tyr Arg Gly Glu Ser Leu Leu Cys Ser Leu Ile Phe 915920 925 Arg Asn Ile Trp Leu Tyr Asn Gln Ile Ala Leu Gln Leu Arg Asn His930 935 940 Ala Leu Cys His Asn Lys Leu Tyr Leu Asp Ile Leu Lys Val LeuLys 945 950 955 960 His Leu Lys Thr Phe Phe Asn Leu Asp Ser Ile Asp MetAla Leu Ser 965 970 975 Leu Tyr Met Asn Leu Pro Met Leu Phe Gly Gly GlyAsp Pro Asn Leu 980 985 990 Leu Tyr Arg Ser Phe Tyr Arg Arg Thr Pro AspPhe Leu Thr Glu Ala 995 1000 1005 Ile Val His Ser Val Phe Val Leu SerTyr Tyr Thr Gly His Asp 1010 1015 1020 Leu Gln Asp Lys Leu Gln Asp LeuPro Asp Asp Arg Leu Asn Lys 1025 1030 1035 Phe Leu Thr Cys Ile Ile ThrPhe Asp Lys Asn Pro Asn Ala Glu 1040 1045 1050 Phe Val Thr Leu Met ArgAsp Pro Gln Ala Leu Gly Ser Glu Arg 1055 1060 1065 Gln Ala Lys Ile ThrSer Glu Ile Asn Arg Leu Ala Val Thr Glu 1070 1075 1080 Val Leu Ser IleAla Pro Asn Lys Ile Phe Ser Lys Ser Ala Gln 1085 1090 1095 His Tyr ThrThr Thr Glu Ile Asp Leu Asn Asp Ile Met Gln Asn 1100 1105 1110 Ile GluPro Thr Tyr Pro His Gly Leu Arg Val Val Tyr Glu Ser 1115 1120 1125 LeuPro Phe Tyr Lys Ala Glu Lys Ile Val Asn Leu Ile Ser Gly 1130 1135 1140Thr Lys Ser Ile Thr Asn Ile Leu Glu Lys Thr Ser Ala Ile Asp 1145 11501155 Thr Thr Asp Ile Asn Arg Ala Thr Asp Met Met Arg Lys Asn Ile 11601165 1170 Thr Leu Leu Ile Arg Ile Leu Pro Leu Asp Cys Asn Lys Asp Lys1175 1180 1185 Arg Glu Leu Leu Ser Leu Glu Asn Leu Ser Ile Thr Glu LeuSer 1190 1195 1200 Lys Tyr Val Arg Glu Arg Ser Trp Ser Leu Ser Asn IleVal Gly 1205 1210 1215 Val Thr Ser Pro Ser Ile Met Phe Thr Met Asp IleLys Tyr Thr 1220 1225 1230 Thr Ser Thr Ile Ala Ser Gly Ile Ile Ile GluLys Tyr Asn Val 1235 1240 1245 Asn Ser Leu Thr Arg Gly Glu Arg Gly ProThr Lys Pro Trp Val 1250 1255 1260 Gly Ser Ser Thr Gln Glu Lys Lys ThrMet Pro Val Tyr Asn Arg 1265 1270 1275 Gln Val Leu Thr Lys Lys Gln ArgAsp Gln Ile Asp Leu Leu Ala 1280 1285 1290 Lys Leu Asp Trp Val Tyr AlaSer Ile Asp Asn Lys Asp Glu Phe 1295 1300 1305 Met Glu Glu Leu Ser ThrGly Thr Leu Gly Leu Ser Tyr Glu Lys 1310 1315 1320 Ala Lys Lys Leu PhePro Gln Tyr Leu Ser Val Asn Tyr Leu His 1325 1330 1335 Arg Leu Thr ValSer Ser Arg Pro Cys Glu Phe Pro Ala Ser Ile 1340 1345 1350 Pro Ala TyrArg Thr Thr Asn Tyr His Phe Asp Thr Ser Pro Ile 1355 1360 1365 Asn HisVal Leu Thr Glu Lys Tyr Gly Asp Glu Asp Ile Asp Ile 1370 1375 1380 ValPhe Gln Asn Cys Ile Ser Phe Gly Leu Ser Leu Met Ser Val 1385 1390 1395Val Glu Gln Phe Thr Asn Ile Cys Pro Asn Arg Ile Ile Leu Ile 1400 14051410 Pro Lys Leu Asn Glu Ile His Leu Met Lys Pro Pro Ile Phe Thr 14151420 1425 Gly Asp Val Asp Ile Ile Lys Leu Lys Gln Val Ile Gln Lys Gln1430 1435 1440 His Met Phe Leu Pro Asp Lys Ile Ser Leu Thr Gln Tyr ValGlu 1445 1450 1455 Leu Phe Leu Ser Asn Lys Ala Leu Lys Ser Gly Ser HisIle Asn 1460 1465 1470 Ser Asn Leu Ile Leu Val His Lys Met Ser Asp TyrPhe His Asn 1475 1480 1485 Asp Tyr Ile Leu Ser Thr Asn Leu Ala Gly HisTrp Ile Leu Ile 1490 1495 1500 Ile Gln Leu Met Lys Asp Ser Lys Gly IlePhe Glu Lys Asp Trp 1505 1510 1515 Gly Glu Gly Tyr Ile Thr Asp His MetPhe Ile Asn Leu Asn Val 1520 1525 1530 Phe Phe Asn Ala Tyr Lys Thr TyrLeu Leu Cys Phe His Lys Gly 1535 1540 1545 Tyr Gly Lys Ala Lys Leu GluCys Asp Met Asn Thr Ser Asp Leu 1550 1555 1560 Leu Cys Val Leu Glu LeuIle Asp Ser Ser Tyr Trp Lys Ser Met 1565 1570 1575 Ser Lys Val Phe LeuGlu Gln Lys Val Ile Lys Tyr Ile Val Asn 1580 1585 1590 Gln Asp Thr SerLeu His Arg Ile Lys Gly Cys His Ser Phe Lys 1595 1600 1605 Leu Trp PheLeu Lys Arg Leu Asn Asn Ala Lys Phe Thr Val Cys 1610 1615 1620 Pro TrpVal Val Asn Ile Asp Tyr His Pro Thr His Met Lys Ala 1625 1630 1635 IleLeu Ser Tyr Ile Asp Leu Val Arg Met Gly Leu Ile Asn Val 1640 1645 1650Asp Lys Leu Thr Ile Lys Asn Lys Asn Lys Phe Asn Asp Glu Phe 1655 16601665 Tyr Thr Ser Asn Leu Phe Tyr Ile Ser Tyr Asn Phe Ser Asp Asn 16701675 1680 Thr His Leu Leu Thr Lys Gln Ile Arg Ile Ala Asn Ser Glu Leu1685 1690 1695 Glu Asn Asn Tyr Asn Lys Leu Tyr His Pro Thr Pro Glu ThrLeu 1700 1705 1710 Glu Asn Met Ser Leu Ile Pro Val Lys Ser Asn Asn SerAsn Lys 1715 1720 1725 Pro Lys Ser Cys Ile Ser Gly Asn Thr Glu Ser MetMet Thr Ser 1730 1735 1740 Thr Phe Ser Asn Lys Met His Ile Lys Ser SerThr Val Thr Thr 1745 1750 1755 Arg Leu Asn Tyr Ser Lys Gln Asp Leu TyrAsn Leu Phe Pro Ile 1760 1765 1770 Val Val Ile Asp Arg Ile Ile Asp HisSer Gly Asn Thr Ala Lys 1775 1780 1785 Ser Asn Gln Leu Tyr Thr Thr ThrSer His Gln Thr Ser Leu Val 1790 1795 1800 Arg Asn Ser Ala Ser Leu TyrCys Met Leu Pro Trp His His Val 1805 1810 1815 Asn Arg Phe Asn Phe ValPhe Ser Ser Thr Gly Cys Lys Ile Ser 1820 1825 1830 Ile Glu Tyr Ile LeuLys Asp Leu Lys Ile Lys Asp Pro Ser Cys 1835 1840 1845 Ile Ala Phe IleGly Glu Gly Ala Gly Asn Leu Leu Leu Arg Thr 1850 1855 1860 Val Val GluLeu His Pro Asp Ile Arg Tyr Ile Tyr Arg Ser Leu 1865 1870 1875 Lys AspCys Asn Asp His Ser Leu Pro Ile Glu Phe Leu Arg Leu 1880 1885 1890 TyrAsn Gly His Ile Asn Ile Asp Tyr Gly Glu Asn Leu Thr Ile 1895 1900 1905Pro Ala Thr Asp Ala Thr Asn Asn Ile His Trp Ser Tyr Leu His 1910 19151920 Ile Lys Phe Ala Glu Pro Ile Ser Ile Phe Val Cys Asp Ala Glu 19251930 1935 Leu Pro Val Thr Ala Asn Trp Ser Lys Ile Ile Ile Glu Trp Ser1940 1945 1950 Lys His Val Arg Lys Cys Lys Tyr Cys Ser Ser Val Asn ArgCys 1955 1960 1965 Ile Leu Ile Ala Lys Tyr His Ala Gln Asp Asp Ile AspPhe Lys 1970 1975 1980 Leu Asp Asn Ile Thr Ile Leu Lys Thr Tyr Val CysLeu Gly Ser 1985 1990 1995 Lys Leu Lys Gly Ser Glu Val Tyr Leu Val LeuThr Ile Gly Pro 2000 2005 2010 Ser Asn Ile Leu Pro Val Phe Asn Val ValGln Asn Ala Lys Leu 2015 2020 2025 Ile Leu Ser Arg Thr Lys Asn Phe IleMet Pro Lys Lys Thr Asp 2030 2035 2040 Lys Glu Ser Ile Asp Ala Asn IleLys Ser Leu Ile Pro Phe Leu 2045 2050 2055 Cys Tyr Pro Ile Thr Lys LysGly Ile Lys Thr Ser Leu Ser Lys 2060 2065 2070 Leu Lys Ser Val Val AsnGly Asp Ile Leu Ser Tyr Ser Ile Ala 2075 2080 2085 Gly Arg Asn Glu ValPhe Ser Asn Lys Leu Ile Asn His Lys His 2090 2095 2100 Met Asn Ile LeuLys Trp Leu Asp His Val Leu Asn Phe Arg Ser 2105 2110 2115 Thr Glu LeuAsn Tyr Asn His Leu Tyr Met Ile Glu Ser Thr Tyr 2120 2125 2130 Pro TyrLeu Ser Glu Leu Leu Asn Ser Leu Thr Thr Asn Glu Leu 2135 2140 2145 LysLys Leu Ile Lys Ile Thr Gly Ser Val Leu Tyr Asn Leu Pro 2150 2155 2160Asn Glu Gln 2165 12 292 PRT respiratory syncytial virus B 9320 12 MetSer Lys His Lys Ser Gln Arg Thr Ala Arg Thr Leu Glu Lys Thr 1 5 10 15Trp Asp Thr Leu Asn His Leu Ile Val Ile Ser Ser Cys Leu Tyr Arg 20 25 30Leu Asn Leu Lys Ser Ile Ala Gln Ile Ala Leu Ser Val Leu Ala Met 35 40 45Ile Ile Ser Thr Ser Leu Ile Ile Ala Ala Ile Ile Phe Ile Ile Ser 50 55 60Ala Asn His Lys Val Thr Leu Thr Thr Val Thr Val Gln Thr Ile Lys 65 70 7580 Asn His Thr Glu Lys Asn Ile Thr Thr Tyr Leu Thr Gln Val Ser Pro 85 9095 Glu Arg Val Ser Ser Ser Ile Gln Pro Thr Thr Thr Ser Pro Ile His 100105 110 Thr Asn Ser Ala Thr Ile Ser Pro Asn Thr Lys Ser Glu Thr His His115 120 125 Thr Thr Thr Gln Ala Lys Ser Arg Ile Thr Thr Ser Thr Gln ThrAsn 130 135 140 Lys Pro Ser Thr Lys Ser Arg Ser Lys Asn Pro Pro Lys LysPro Lys 145 150 155 160 Asp Asp Tyr His Phe Glu Val Phe Asn Phe Val ProCys Ser Ile Cys 165 170 175 Gly Asn Asn Gln Leu Cys Lys Ser Ile Cys LysThr Ile Pro Ser Asn 180 185 190 Lys Pro Lys Lys Lys Pro Thr Ile Lys ProThr Asn Lys Pro Thr Val 195 200 205 Lys Thr Thr Asn Lys Arg Asp Pro LysThr Pro Ala Lys Met Met Lys 210 215 220 Lys Glu Thr Thr Thr Asn Pro ThrLys Lys Pro Thr Leu Lys Thr Thr 225 230 235 240 Glu Gly Asp Thr Ser ThrSer Gln Ser Thr Val Leu Asp Thr Thr Thr 245 250 255 Ser Lys His Thr IleGln Gln Gln Ser Leu His Ser Ile Thr Ser Glu 260 265 270 Asn Thr Pro AsnSer Thr Gln Ile Pro Thr Ala Thr Glu Ala Ser Thr 275 280 285 Ser Asn SerThr 290 13 15225 DNA respiratory syncytial virus B 1 13 acgcgaaaaaatgcgtacta caaacttgca cattcggaaa aaatggggca aataagaatt 60 tgataagtgctatttaagtc taaccttttc aatcagaaat ggggtgcaat tcactgagca 120 tgataaaggttagattacaa aatttatttg acaatgacga agtagcattg ttaaaaataa 180 catgttatactgacaaatta attcttctga ccaatgcatt agccaaagca gcaatacata 240 caattaaattaaacggtata gtttttatac atgttataac aagcagtgaa gtgtgccctg 300 ataacaacattgtagtaaaa tctaacttta caacaatgcc aatattacaa aacggaggat 360 acatatgggaattgattgag ttgacacact gctctcaatt aaacggtcta atggatgata 420 attgtgaaatcaaattttct aaaagactaa gtgactcagt aatgactaat tatatgaatc 480 aaatatctgatttacttggg cttgatctca attcatgaat tatgtttagt ctaactcaat 540 agacatgtgtttattaccat tttagttaat ataaaaactc atcaaaggga aatggggcaa 600 ataaactcacctaatcaatc aaactatgag cactacaaat gacaacacta ctatgcaaag 660 attaatgatcacggacatga gacccctgtc gatggattca ataataacat ctctcaccaa 720 agaaatcatcacacacaaat tcatatactt gataaacaat gaatgtattg taagaaaact 780 tgatgaaagacaagctacat ttacattctt agtcaattat gagatgaagc tactgcacaa 840 agtagggagtaccaaataca agaaatacac tgaatataat acaaaatatg gcactttccc 900 catgcctatatttatcaatc atggcgggtt tctagaatgt attggcatta agcctacaaa 960 acacactcctataatataca aatatgacct caacccgtaa attccaacaa aaaaaaccaa 1020 cccaaccaaaccaagctatt cctcaaacaa caatgctcaa tagttaagaa ggagctaatc 1080 cgttttagtaattaaaaata aaagtaaagc caataacata aattggggca aatacaaaga 1140 tggctcttagcaaagtcaag ttaaatgata cattaaataa ggatcagctg ctgtcatcca 1200 gcaaatacactattcaacgt agtacaggag ataatattga cactcccaat tatgatgtgc 1260 aaaaacacctaaacaaacta tgtggtatgc tattaatcac tgaagatgca aatcataaat 1320 tcacaggattaataggtatg ttatatgcta tgtccaggtt aggaagggaa gacactataa 1380 agatacttaaagatgctgga tatcatgtta aagctaatgg agtagatata acaacatatc 1440 gtcaagatataaatggaaag gaaatgaaat tcgaagtatt aacattatca agcttgacat 1500 cagaaatacaagtcaatatt gagatagaat ctagaaaatc ctacaaaaaa atgctaaaag 1560 agatgggagaagtggctcca gaatataggc atgattctcc agactgtggg atgataatac 1620 tgtgtatagcagcacttgta ataaccaaat tagcagcagg agacagatca ggtcttacag 1680 cagtaattaggagggcaaac aatgtcttaa aaaatgaaat aaaacgctac aagggtctca 1740 taccaaaggatatagctaac agtttttatg aagtgtttga aaaacaccct catcttatag 1800 atgtttttgtgcactttggc attgcacaat catcaacaag agggggtagt agagttgaag 1860 gaatctttgcaggattgttt atgaatgcct atggttcagg gcaagtaatg ctaagatggg 1920 gagttttagccaaatctgta aaaaatatca tgctaggtca tgctagtgtc caggcagaaa 1980 tggagcaagttgtggaagtc tatgagtatg cacagaagtt gggaggagaa gctggattct 2040 accatatattgaacaatcca aaagcatcat tgctgtcatt aactcaattt cctaacttct 2100 caagtgtggtcctaggcaat gcagcaggtc taggcataat gggagagtat agaggtacgc 2160 caagaaaccaggatctttat gatgcagcca aagcatatgc agagcaactc aaagaaaatg 2220 gagtaataaactacagtgta ttagacttaa cagcagaaga attggaagcc ataaagaatc 2280 aactcaaccctaaagaagat gatgtagagc tttaagttaa caaaaaatac ggggcaaata 2340 agtcaacatggagaagtttg cacctgaatt tcatggagaa gatgcaaata acaaagctac 2400 caaattcctagaatcaataa agggcaagtt cgcatcatcc aaagatccta agaagaaaga 2460 tagcataatatctgttaact caatagatat agaagtaacc aaagagagcc cgataacatc 2520 tggcaccaacatcatcaatc caacaagtga agccgacagt accccagaaa ccaaagccaa 2580 ctacccaagaaaacccctag taagcttcaa agaagatctc accccaagtg acaacccttt 2640 ttctaagttgtacaaagaaa caatagaaac atttgataac aatgaagaag aatctagcta 2700 ctcatatgaagagataaatg atcaaacaaa tgacaacatt acagcaagac tagatagaat 2760 tgatgaaaaattaagtgaaa tattaggaat gctccataca ttagtagttg caagtgcagg 2820 acccacttcagctcgcgatg gaataagaga tgctatggtt ggtctgagag aagaaatgat 2880 agaaaaaataagagcggaag cattaatgac caatgatagg ttagaggcta tggcaagact 2940 taggaatgaggaaagcgaaa aaatggcaaa agacacctca gatgaagtgc ctcttaatcc 3000 aacttccaaaaaattgagtg acttgttgga agacaacgat agtgacaatg atctgtcact 3060 tgatgatttttgatcagtga tcaactcact cagcaatcaa caacatcaat aaaacagaca 3120 tcaatccattgaatcaactg ccagaccgaa caaacaaatg tccgtcagcg gaaccaccaa 3180 ccaatcaatcaaccaactga tccatcagca acctgacgaa attaacaata tagtaacaaa 3240 aaaagaacaagatggggcaa atatggaaac atacgtgaac aagcttcacg aaggctccac 3300 atacacagcagctgttcagt acaatgttct agaaaaagat gatgatcctg catcactaac 3360 aatatgggtgcctatgttcc agtcatctgt accagcagac ttgctcataa aagaacttgc 3420 aagcatcaacatactagtga agcagatctc tacgcccaaa ggaccttcac tacgagtcac 3480 gattaactcaagaagtgctg tgctggctca aatgcctagt aatttcatca taagcgcaaa 3540 tgtatcattagatgaaagaa gcaaattagc atatgatgta actacacctt gtgaaatcaa 3600 agcatgcagtctaacatgct taaaagtgaa aagtatgtta actacagtca aagatcttac 3660 catgaagacattcaacccca ctcatgagat cattgctcta tgtgaatttg aaaatattat 3720 gacatcaaaaagagtaataa taccaaccta tctaagacca attagtgtca aaaacaagga 3780 tctgaactcactagaaaaca tagcaaccac cgaattcaaa aatgctatca ccaatgcgaa 3840 aattattccctatgctggat tagtattagt tatcacagtt actgacaata aaggagcatt 3900 caaatatatcaagccacaga gtcaatttat agtagatctt ggtgcctacc tagaaaaaga 3960 gagcatatattatgtgacta ctaattggaa gcatacagct acacgttttt caatcaaacc 4020 actagaggattaaatttaat tatcaacact gaatgacagg tccacatata tcctcaaact 4080 acacactatatccaaacatc atgaacatct acactacaca cttcatcaca caaaccaatc 4140 ccactcaaaatccaaaatca ctaccagcca ctatctgcta gacctagagt gcgaataggt 4200 aaataaaaccaaaatatggg gtaaatagac attagttaga gttcaatcaa tctcaacaac 4260 catttataccgccaattcaa tacatatact ataaatctta aaatgggaaa tacatccatc 4320 acaatagaattcacaagcaa attttggccc tattttacac taatacatat gatcttaact 4380 ctaatctctttactaattat aatcactatt atgattgcaa tactaaataa gctaagtgaa 4440 cataaaacattctgtaacaa tactcttgaa ctaggacaga tgcatcaaat caacacatag 4500 tgctctaccatcatgctgtg tcaaattata atcctgtata tataaacaaa caaatccaat 4560 cttctcacagagtcatggtg tcgcaaaacc acgccaacta tcatggtagc atagagtagt 4620 tatttaaaaattaacataat gatgaattat tagtatggga tcaaaaacaa cattggggca 4680 aatgcaaccatgtccaaaca caagaatcaa cgcactgcca ggactctaga aaagacctgg 4740 gatactctcaatcatctaat tgtaatatcc tcttgtttat acagattaaa tttaaaatct 4800 atagcacaaatagcactatc agttctggca atgataatct caacctctct cataattgca 4860 gccataatattcatcatctc tgccaatcac aaagttacac taacaacggt cacagttcaa 4920 acaataaaaaaccacactga aaaaaacatc accacctacc ttactcaagt cccaccagaa 4980 agggttagctcatccaaaca acctacaacc acatcaccaa tccacacaaa ttcagccaca 5040 acatcacccaacacaaagtc agaaacacac cacacaacag cacaaaccaa aggcagaacc 5100 accacctcaacacagaccaa caagccgagc acaaaaccac gcctaaaaaa tccaccaaaa 5160 aaaccaaaagatgattacca ttttgaagtg ttcaacttcg ttccctgtag tatatgtggc 5220 aacaatcaactttgcaaatc catctgtaaa acaataccaa gcaacaaacc aaagaagaaa 5280 ccaaccatcaaacccacaaa caaaccaacc accaaaacca caaacaaaag agacccaaaa 5340 acaccagccaaaacgacgaa aaaagaaact accaccaacc caacaaaaaa accaaccctc 5400 acgaccacagaaagagacac cagcacctca caatccactg tgctcgacac aaccacatta 5460 gaacacacaatccaacagca atccctccac tcaaccaccc ccgaaaacac acccaactcc 5520 acacaaacacccacagcatc cgagccctct acatcaaatt ccacccaaaa tacccaatca 5580 catgcttagttattcaaaaa ctacatctta gcagaaaacc gtgacctatc aagcaagaac 5640 gaaattaaacctggggcaaa taaccatgga gctgctgatc cacaggttaa gtgcaatctt 5700 cctaactcttgctattaatg cattgtacct cacctcaagt cagaacataa ctgaggagtt 5760 ttaccaatcgacatgtagtg cagttagcag aggttatttt agtgctttaa gaacaggttg 5820 gtataccagtgtcataacaa tagaattaag taatataaaa gaaaccaaat gcaatggaac 5880 tgacactaaagtaaaactta taaaacaaga attagataag tataagaatg cagtgacaga 5940 attacagctacttatgcaaa acacaccagc tgccaacaac cgggccagaa gagaagcacc 6000 acagtatatgaactatacaa tcaataccac taaaaaccta aatgtatcaa taagcaagaa 6060 gaggaaacgaagatttctgg gcttcttgtt aggtgtagga tctgcaatag caagtggtat 6120 agctgtatccaaagttctac accttgaagg agaagtgaac aagatcaaaa atgctttgtt 6180 atctacaaacaaagctgtag tcagtctatc aaatggggtc agtgttttaa ccagcaaagt 6240 gttagatctcaagaattaca taaataacca attattaccc atagtaaatc aacagagctg 6300 tcgcatctccaacattgaaa cagttataga attccagcag aagaacagca gattgttgga 6360 aatcaacagagaattcagtg tcaatgcagg tgtaacaaca cctttaagca cttacatgtt 6420 aacaaacagtgagttactat cattgatcaa tgatatgcct ataacaaatg atcagaaaaa 6480 attaatgtcaagcaatgttc agatagtaag gcaacaaagt tattctatca tgtctataat 6540 aaaggaagaagtccttgcat atgttgtaca gctacctatc tatggtgtaa tagatacacc 6600 ttgctggaaattacacacat cacctctatg caccaccaac atcaaagaag gatcaaatat 6660 ttgtttaacaaggactgata gaggatggta ttgtgataat gcaggatcag tatccttctt 6720 tccacaggctgacacttgta aagtacagtc caatcgagta ttttgtgaca ctatgaacag 6780 tttgacattaccaagtgaag tcagcctttg taacactgac atattcaatt ccaagtatga 6840 ctgcaaaattatgacatcaa aaacagacat aagcagctca gtaattactt ctcttggagc 6900 tatagtgtcatgctatggta aaactaaatg cactgcatcc aacaaaaatc gtgggattat 6960 aaagacattttctaatggtt gtgactatgt gtcaaacaaa ggagtagata ctgtgtcagt 7020 gggcaacactttatactatg taaacaagct ggaaggcaag aacctttatg taaaagggga 7080 acctataataaattactatg accctctagt gtttccttct gatgagtttg atgcatcaat 7140 atctcaagtcaatgaaaaaa tcaatcaaag tttagctttt attcgtagat ctgatgaatt 7200 actacataatgtaaatactg gcaaatctac tacaaatatt atgataacta caattattat 7260 agtaatcattgtagtattgt tatcattaat agctattggt ttgctgttgt attgcaaagc 7320 caaaaacacaccagttacac taagcaaaga ccaactaagt ggaatcaata atattgcatt 7380 cagcaaatagacaaaaaacc acctgatcat gtttcaacaa cagtctgctg atcaccaatc 7440 ccaaatcaacccataacaaa cacttcaaca tcacagtaca ggctgaatca tttcttcaca 7500 tcatgctacccacacaacta agctagatcc ttaactcata gttacataaa aacctcaagt 7560 atcacaatcaaacactaaat caacacatca ttcacaaaat taacagctgg ggcaaatatg 7620 tcgcgaagaaatccttgtaa atttgagatt agaggtcatt gcttgaatgg tagaagatgt 7680 cactacagtcataattactt tgaatggcct cctcatgcct tactagtgag gcaaaacttc 7740 atgttaaacaagatactcaa gtcaatggac aaaagcatag acactttgtc tgaaataagt 7800 ggagctgctgaactggacag aacagaagaa tatgctcttg gtatagttgg agtgctagag 7860 agttacataggatctataaa caacataaca aaacaatcag catgtgttgc tatgagtaaa 7920 cttcttattgagatcaatag tgatgacatt aaaaagctga gagataatga agaacccaat 7980 tcacctaagataagagtgta caatactgtt atatcataca ttgagagcaa tagaaaaaac 8040 aacaagcaaacaatccatct gctcaaaaga ctaccagcag acgtgctgaa gaagacaata 8100 aaaaacacattagatatcca caaaagcata atcataagca acccaaaaga gtcaaccgtg 8160 aatgatcaaaatgaccaaac caaaaataat gatattaccg gataaatatc cttgtagtat 8220 atcatccatattgatttcaa gtgaaagcat gattgctaca ttcaatcata aaaacatatt 8280 acaatttaaccataaccatt tggataacca ccagcgttta ttaaataata tatttgatga 8340 aattcattggacacctaaaa acttattaga tgccactcaa caatttctcc aacatcttaa 8400 catccctgaagatatatata caatatatat attagtgtca taatgcttgg ccataacgat 8460 tctatatcatccaaccataa aactatctta ataaggttat gggacaaaat ggatcccatt 8520 attaatggaaactctgctaa tgtgtatcta actgatagtt atttaaaagg tgttatctct 8580 ttttcagaatgtaatgcttt agggagttac ctttttaacg gcccttatct caaaaatgat 8640 tacaccaacttaattagtag acaaagtcca ctactagagc atatgaatct taaaaaacta 8700 actataacacagtcattaat atctagatat cataaaggtg aactgaaatt agaagaacca 8760 acttatttccagtcattact tatgacatat aaaagcatgt cctcgtctga acaaattgct 8820 acaactaacttacttaaaaa aataatacga agagctatag aaataagtga tgtaaaggtg 8880 tacgccatcttgaataaact aggactaaag gaaaaggaca gagttaagcc caacaataat 8940 tcaggtgatgaaaactcagt acttacaact ataattaaag atgatatact ttcggctgtg 9000 gaaagcaatcaatcatatac aaattcagac aaaaatcact cagtaaatca aaatatcact 9060 atcaaaacaacactcttgaa aaaattgatg tgttcaatgc aacatcctcc atcatggtta 9120 atacactggttcaatttata tacaaaatta aataacatat taacacaata tcgatcaaat 9180 gaggtaaaaagtcatgggtt tatattaata gataatcaaa ctttaagtgg ttttcagttt 9240 attttaaatcaatatggttg tatcgtttat cataaaggac tcaaaaaaat cacaactact 9300 acttacaatcaatttttaac atggaaagac atcagcctta gcagattaaa tgtttgctta 9360 attacttggataagtaattg tttgaataca ttaaataaaa gcttagggct gagatgtgga 9420 ttcaataatgttgtgttatc acaattattt ctttatggag attgtatact gaaattattt 9480 cataatgaaggcttctacat aataaaagaa gtagagggat ttattatgtc tttaattcta 9540 aacataacagaagaagatca atttaggaaa cgattttata atagcatgct aaataacatc 9600 acagatgcagctattaaggc tcaaaagaac ctactatcaa gggtatgtca cactttatta 9660 gacaagacagtgtctgataa tatcataaat ggtaaatgga taatcctatt aagtaaattt 9720 cttaaattgattaagcttgc aggtgataat aatctcaata atttgagtga gctatatttt 9780 ctcttcagaatctttggaca tccaatggtt gatgaaagac aagcaatgga tgctgtaaga 9840 attaactgtaatgaaactaa gttctactta ttaagtagtc taagtacgtt aagaggtgct 9900 ttcatttatagaatcataaa agggtttgta aatacctaca acagatggcc cactttaagg 9960 aatgctattgtcctacctct aagatggtta aactattata aacttaatac ttatccatct 10020 ctacttgaaatcacagaaaa tgatttgatt attttatcag gattgcggtt ctatcgtgaa 10080 tttcatctgcctaaaaaagt ggatcttgaa atgataataa atgacaaagc catttcacct 10140 ccaaaagatctaatatggac tagttttcct agaaattaca tgccatcaca tatacaaaat 10200 tatatagaacatgaaaagtt gaagttctct gaaagcgaca gatcaagaag agtactagag 10260 tattacttgagagataataa attcaatgaa tgcgatctat acaattgtgt agtcaatcaa 10320 agctatctcaacaactctaa tcacgtggta tcactaactg gtaaagaaag agagctcagt 10380 gtaggtagaatgtttgctat gcaaccaggt atgtttaggc aaatccaaat cttagcagag 10440 aaaatgatagccgaaaatat tttacaattc ttccctgaga gtttgacaag atatggtgat 10500 ctagagcttcaaaagatatt agaattaaaa gcaggaataa gcaacaagtc aaatcgttat 10560 aatgataactacaacaatta tatcagtaaa tgttctatca ttacagatct tagcaaattc 10620 aatcaagcatttagatatga aacatcatgt atctgcagtg atgtattaga tgaactgcat 10680 ggagtacaatctctgttctc ttggttgcat ttaacaatac ctcttgtcac aataatatgt 10740 acatatagacatgcacctcc tttcataaag gatcatgttg ttaatcttaa tgaagttgat 10800 gaacaaagtggattatacag atatcatatg ggtggtattg agggctggtg tcaaaaactg 10860 tggaccattgaagctatatc attattagat ctaatatctc tcaaagggaa attctctatc 10920 acagctctgataaatggtga taatcagtca attgatataa gtaaaccagt tagacttata 10980 gagggtcagacccatgctca agcagattat ttgttagcat taaatagcct taaattgcta 11040 tataaagagtatgcaggtat aggccataag cttaagggaa cagagaccta tatatcccga 11100 gatatgcagttcatgagcaa aacaatccag cacaatggag tgtactatcc agccagtatc 11160 aaaaaagtcctgagagtagg tccatggata aatacaatac ttgatgattt taaagttagt 11220 ttagaatctataggtagctt aacacaggag ttagaataca gaggggaaag cttattatgc 11280 agtttaatatttaggaacat ttggttatac aatcaaattg ctttgcaact ccgaaatcat 11340 gcattatgtaacaataagct atatttagat atattgaaag tattaaaaca cttaaaaact 11400 ttttttaatcttgatagtat cgatatggcg ttatcattgt atatgaattt gcctatgctg 11460 tttggtggtggtgatcctaa tttgttatat cgaagctttt ataggagaac tccagacttc 11520 cttacagaagctatagtaca ttcagtgttt gtgttgagct attatactgg tcacgattta 11580 caagataagctccaggatct tccagatgat agactgaaca aattcttgac atgtgtcatc 11640 acattcgataaaaatcccaa tgccgagttt gtaacattga tgagggatcc acaggcgtta 11700 gggtctgaaaggcaagctaa aattactagt gagattaata gattagcagt aacagaagtc 11760 ttaagtatagctccaaacaa aatattttct aaaagtgcac aacattatac taccactgag 11820 attgatctaaatgacattat gcaaaatata gaaccaactt accctcatgg attaagagtt 11880 gtttatgaaagtctaccttt ttataaagca gaaaaaatag ttaatcttat atcaggaaca 11940 aaatccataactaatatact tgaaaaaaca tcagcaatag atacaactga tattaatagg 12000 gctactgatatgatgaggaa aaatataact ttacttataa ggatacttcc actagattgt 12060 aacaaagacaaaagagagtt attaagttta gaaaatctta gtataactga attaagcaag 12120 tatgtaagagaaagatcttg gtcattatcc aatatagtag gagtaacatc gccaagtatt 12180 atgttcacaatggacattaa atatacaact agcactatag ccagtggtat aattatagaa 12240 aaatataatgttaatagttt aactcgtggt gaaagaggac ctactaagcc atgggtaggt 12300 tcatctacgcaggagaaaaa aacaatgcca gtgtacaata gacaagtttt aaccaaaaag 12360 caaagagaccaaatagattt attagcaaaa ttagactggg tatatgcatc catagacaac 12420 aaagatgaattcatggaaga actgagtact ggaacacttg gactgtcata tgaaaaagcc 12480 aaaaagttgtttccacaata tctaagtgtc aattatttac accgtttaac agtcagtagt 12540 agaccatgtgaattccctgc atcaatacca gcttatagaa caacaaatta tcatttcgat 12600 actagtcctatcaatcatgt attaacagaa aagtatggag atgaagatat cgacattgtg 12660 tttcaaaattgcataagttt tggtcttagc ctgatgtcgg ttgtggaaca attcacaaac 12720 atatgtcctaatagaattat tctcataccg aagctgaatg agatacattt gatgaaacct 12780 cctatatttacaggagatgt tgatatcatc aagttgaagc aagtgataca aaaacagcat 12840 atgttcctaccagataaaat aagtttaacc caatatgtag aattattcct aagtaacaaa 12900 gcacttaaatctggatctaa catcaattct aatttaatat tagtacataa aatgtctgat 12960 tattttcataatgcttatat tttaagtact aatttagctg gacattggat tctaattatt 13020 caacttatgaaagattcaaa aggtattttt gaaaaagatt ggggagaggg gtacataact 13080 gatcatatgttcattaattt gaatgttttc tttaatgctt ataagactta tttgctatgt 13140 tttcataaaggttatggtaa agcaaaatta gaatgtgata tgaacacttc agatcttctt 13200 tgtgttttggagttaataga cagtagctac tggaaatcta tgtctaaagt tttcctagaa 13260 caaaaagtcataaaatacat agtcaatcaa gacacaagtt tgcatagaat aaaaggctgt 13320 cacagttttaagttgtggtt tttaaaacgc cttaataatg ctaaatttac cgtatgccct 13380 tgggttgttaacatagatta tcacccaaca catatgaaag ctatattatc ttacatagat 13440 ttagttagaatggggttaat aaatgtagat aaattaacca ttaaaaataa aaacaaattc 13500 aatgatgaattttacacatc aaatctcttt tacattagtt ataacttttc agacaacact 13560 catttgctaacaaaacaaat aagaattgct aattcagaat tagaagataa ttataacaaa 13620 ctatatcacccaaccccaga aactttagaa aatatatcat taattcctgt taaaagtaat 13680 aatagtaacaaacctaaatt ttgtataagt ggaaataccg aatctataat gatgtcaaca 13740 ttctctaataaaatgcatat taaatcttcc actgttacca caagattcaa ttatagcaaa 13800 caagacttgtacaatttatt tccaaatgtt gtgatagaca ggattataga tcattcaggt 13860 aatacagcaaaatctaacca actttacatc accacttcac atcagacatc tttagtaagg 13920 aatagtgcatcactttattg catgcttcct tggcatcatg tcaatagatt taactttgta 13980 tttagttccacaggatgcaa gatcagtata gagtatattt taaaagatct taagattaag 14040 gaccccagttgtatagcatt cataggtgaa ggagctggta acttattatt acgtacggta 14100 gtagaacttcatccagacat aagatacatt tacagaagtt taaaagattg caatgatcat 14160 agtttacctattgaatttct aagattatac aacgggcata taaacataga ttatggtgag 14220 aatttaaccattcctgctac agatgcaact aataacattc attggtctta tttacatata 14280 aaatttgcagaacctattag catctttgtc tgcgatgctg aattacctgt tacagccaat 14340 tggagtaaaattataattga atggagtaag catgtaagaa agtgcaagta ctgttcttct 14400 gtaaatagatgcattttaat cgcaaaatat catgctcaag atgatattga tttcaaatta 14460 gataacattactatattaaa aacttacgtg tgcctaggta gcaagttaaa aggatctgaa 14520 gtttacttagtccttacaat aggccctgca aatatacttc ctgtttttga tgttgtgcaa 14580 aatgctaaattgattttttc aagaactaaa aatttcatta tgcctaaaaa aactgacaag 14640 gaatctatcgatgcaaatat taaaagctta atacctttcc tttgttaccc tataacaaaa 14700 aaaggaattaagacttcatt gtcaaaattg aagagtgtag ttaatgggga tatattatca 14760 tattctatagctggacgtaa tgaagtattc agcaacaagc ttataaacca caagcatatg 14820 aatatcctaaaatggctaga tcatgtttta aattttagat cagctgaact taattacaat 14880 catttatacatgatagagtc cacatatcct tacttaagtg aattgttaaa tagtttaaca 14940 accaatgagctcaagaaact gattaaaata acaggtagtg tactatacaa ccttcccaac 15000 gaacagtaacttaaaatatc attaacaagt ttggtcaaat ttagatgcta acacatcatt 15060 atattatagttattaaaaaa tatgcaaact tttcaataat ttagcttact gattccaaaa 15120 ttatcattttatttttaagg ggttgaataa aagtctaaaa ctaacaatga tacatgtgca 15180 tttacaacacaacgagacat tagtttttga cacttttttt ctcgt 15225 14 868 DNA respiratorysyncytial virus B 9320 14 agtcaacgca ctgccaggac tctagaaaag acctgggatactcttaatca tctaattgta 60 atatcctctt gtttatacag actaaaccta aaatctatagcacaaatagc actatcagtt 120 ttggcaatga taatctcaac ctctctcata attgcagccataatattcat catctctgcc 180 aatcacaaag ttacactaac aacggttaca gttcaaacaataaaaaacca cactgaaaaa 240 aacatcacca cctaccttac tcaagtctca ccagaaagggttagctcatc catacaacct 300 acaaccacat caccaatcca cacaaattca gctacaatatcaccaaatac aaaatcagaa 360 acacaccata caacaacaca agccaaaagc agaatcaccacttcaacaca gaccaacaag 420 ccaagcacaa aatcacgttc aaaaaatcca ccaaaaaaaccaaaagatga ttaccatttt 480 gaagtgttca attttgttcc ctgtagtata tgtggcaacaatcaactttg caaatccatc 540 tgcaaaacaa taccaagcaa caaaccaaag aaaaaaccaaccatcaaacc cacaaacaaa 600 ccaaccgtca aaaccacaaa caaaagagac ccaaaaacaccagccaaaat gatgaaaaaa 660 gaaaccacca ccaacccaac aaaaaaacca accctcaagaccacagaagg agacaccagc 720 acctcacaat ccactgtgct cgacacaacc acatcaaaacacacaatcca acagcaatcc 780 ctccactcaa tcacctccga aaacacaccc aactccacacaaatacccac agcaaccgag 840 gcctccacat caaattctac ttaaaaaa 868 15 218 DNArespiratory syncytial virus B 9320 15 attggcatta agcctacaaa acacactcctataatataca aatatgacct caacccgtaa 60 attccaacaa aaaactaacc catccaaactaagctattcc ttaaataaca gtgctcaaca 120 gttaagaagg ggctaatcca ttttagtaattaaaaataaa ggtaaagcca ataacataaa 180 ttggggcaaa tacaaagatg gctcttagcaaagtcaag 218 16 35 DNA Artificial oligonucleotide primer; BglIIsite, RSVB 9320 G 16 gatatcaaga tctacaataa cattggggca aatgc 35 17 31 DNAArtificial oligonucleotide primer; BglIIsite, RSV B 9320 G 17 gctaagagatctttttgaat aactaagcat g 31 18 36 DNA Artificial oligonucleotide primer;BamHIsite, RSV B 9320 18 atcaggatcc acaataacat tggggcaaat gcaacc 36 1936 DNA Artificial oligonucleotide primer; BamHI site, RSV 9320 G 19ctggcattcg gatccgtttt atgtaactat gagttg 36 20 27 DNA Artificialoligonucleotide primer 20 gatcccatgg ctcttagcaa agtcaag 27 21 31 DNAArtificial oligonucleotide primer 21 gtacggatcc gttgacttat ttgccccgta t31 22 25 DNA Artificial oligonucleotide primer 22 gatcccatgg agaagtttgcacctg 25 23 28 DNA Artificial oligonucleotide primer 23 gtacggatcctgagtgagtt gatcactg 28 24 28 DNA Artificial oligonucleotide primer 24gcttggccat aacgattcta tatcatcc 28 25 26 DNA Artificial oligonucleotideprimer 25 ggtagtataa tgttgtgcac ttttag 26 26 25 DNA Artificialoligonucleotide primer 26 ggtcacgatt tacaagataa gctcc 25 27 30 DNAArtificial oligonucleotide primer 27 cagatccttt taacttgcta cctaggcaca 3028 23 DNA Artificial oligonucleotide primer 28 cttacgtgtg cctaggtagc aag23 29 33 DNA Artificial oligonucleotide primer 29 acgagaaaaa aagtgtcaaaaactaatgtc tcg 33 30 42 DNA Artificial oligonucleotide primer 30gtttttgaca ctttttttct cgtggccggc atggtcccag cc 42 31 33 DNA Artificialoligonucleotide primer 31 gatctagagc tccaagcttg cggccgcgtc gac 33 32 46DNA Artificial oligonucleotide primer 32 gggtaccccc gggtaatacgactcactata gggacgggaa aaaatg 46 33 24 DNA Artificial oligonucleotideprimer 33 gttaacttag agctctacat catc 24 34 24 DNA Artificialoligonucleotide primer 34 gtgtggtcct aggcaatgca gcag 24 35 29 DNAArtificial oligonucleotide primer 35 gacacagcat gatggtagag ctctatgtg 2936 28 DNA Artificial oligonucleotide primer 36 gctaagtgaa cataaaacattctgtaac 28 37 26 DNA Artificial oligonucleotide primer 37 ccattaataatgggatccat tttgtc 26 38 29 DNA Artificial oligonucleotide primer 38cacatagagc tctaccatca tgctgtgtc 29 39 27 DNA Artificial oligonucleotideprimer 39 cattaatgag ggacccacag gctttag 27 40 27 DNA Artificialoligonucleotide primer 40 ctaaagcctg tgggtccctc attaatg 27 41 32 DNAArtificial oligonucleotide primer 41 catggttaat acactggttc aatttatata ca32 42 32 DNA Artificial oligonucleotide primer 42 tgtatataaa ttgaaccagtgtattaacca tg 32 43 41 DNA Artificial oligonucleotide primer 43gtcttaaaaa acgaaataaa acgctacaag ggcctcatac c 41 44 41 DNA Artificialoligonucleotide primer 44 ggtatgaggc ccttgtagcg ttttatttcg ttttttaaga c41 45 24 DNA Artificial oligonucleotide primer 45 gatgatgtag agctttaagttaac 24 46 24 DNA Artificial oligonucleotide primer 46 gttaacttaaagctctacat catc 24 47 44 DNA Artificial oligonucleotide primer 47ctaactggta aagaaagaga gcttagtgta ggtagaatgt ttgc 44 48 44 DNA Artificialoligonucleotide primer 48 gcaaacattc tacctacact aagctctctt tctttaccagttag 44 49 40 DNA Artificial oligonucleotide primer 49 gtttaacaaccaatgagctt aaaaagctga ttaaaattac 40 50 40 DNA Artificial oligonucleotideprimer 50 gtaattttaa tcagcttttt aagctcattg gttgttaaac 40 51 23 DNAArtificial oligonucleotide primer 51 cggtctaatg gatgataatt gtg 23 52 23DNA Artificial oligonucleotide primer 52 atgaagctac tgcacaaagt agg 23 5327 DNA Artificial oligonucleotide primer 53 gtaatcatct tttggtttttttggtgg 27 54 33 DNA Artificial oligonucleotide primer 54 ccaaccatcaaacccacaaa caaaccaacc gtc 33

1. An isolated or recombinant nucleic acid comprising a polynucleotidesequence selected from the group consisting of: (a) SEQ ID NO:1 or acomplementary polynucleotide sequence thereof; (b) a polynucleotidesequence that is greater than 97.8% identical to SEQ ID NO:1 or acomplementary polynucleotide sequence thereof, as determined by BLASTNusing default parameters; (c) a polynucleotide sequence comprising atleast one unique polynucleotide subsequence comprising at least 10contiguous nucleotides of SEQ ID NO:1 or a complementary polynucleotidesequence thereof, with the proviso that the unique polynucleotidesubsequence includes at least one subsequence not included in SEQ IDNOs:14-19 or a complementary polynucleotide sequence thereof; and, (d) apolynucleotide sequence encoding an amino acid sequence or uniquesubsequence selected from the group consisting of SEQ ID NOs:2-11 or anartificial conservative variation thereof.
 2. The nucleic acid of claim1, wherein the nucleic acid is selected from the group consisting of aDNA, a cDNA, an RNA, and an artificial nucleic acid.
 3. (Canceled) 4.The nucleic acid of claim 1, wherein the polynucleotide sequence of (b)is at least 98.5% identical to SEQ ID NO:1 or a complementarypolynucleotide sequence thereof, as determined by BLASTN using defaultparameters.
 5. (Canceled)
 6. The nucleic acid of claim 1, comprising atleast one artificially mutated nucleotide or comprising at least oneartificially mutated nucleotide which comprises one or more of: adeleted nucleotide, an inserted nucleotide, or a substituted nucleotide.7-9. (Canceled)
 10. The nucleic acid of claim 6, wherein at least onepolypeptide encoded by the nucleic acid comprises at least one deleted,inserted, or substituted amino acid residue.
 11. The nucleic acid ofclaim 10, wherein the polypeptide comprises at least one conservativelysubstituted amino acid residue.
 12. The nucleic acid of claim 6, whereinthe at least one artificially mutated nucleotide is located in the openreading frame encoding the polypeptide of SEQ ID NO:12.
 13. (Canceled)14. The nucleic acid of claim 6, wherein the open reading frame encodingthe polypeptide of SEQ ID NO:12 is deleted, or wherein the open readingframe encoding the polypeptide of SEQ ID NO:10 is deleted.
 15. Thenucleic acid of claim 12, wherein the at least one artificially mutatednucleotide comprises a deletion, and wherein the nucleotides encodingamino acid residues 164-197 of SEQ ID NO:12 are deleted.
 16. The nucleicacid of claim 6, wherein the at least one artificially mutatednucleotide is located in the open reading frame encoding the polypeptideof SEQ ID NO:10. 17-18. (Canceled)
 19. The nucleic acid of claim 16,wherein at least one of the nucleotides encoding amino acid residue 1,amino acid residue 4, amino acid residue 10, or a combination thereof,of SEQ ID NO:10 is mutated.
 20. The nucleic acid of claim 1, wherein theunique polynucleotide subsequence of (c) encodes at least 20, at least50, at least 100, or at least 200 contiguous amino acid residues of anyone of SEQ ID NOs:2-12; wherein the unique polynucleotide subsequence of(d) comprises at least one complete open reading frame; wherein theunique polynucleotide subsequence of (d) comprises at least one completeopen reading frame which encodes a polypeptide selected from the groupconsisting of SEQ ID NOs:2-12; or comprising a plurality of completeopen reading frames. 21-26. (Canceled)
 27. The nucleic acid of claim 1,wherein the nucleic acid of (c) further comprises at least onepolynucleotide subsequence from a different strain of virus, at leastone polynucleotide subsequence from a different strain of human RSV, orat least one polynucleotide subsequence from a different species ofvirus. 28-34. (Canceled)
 35. A recombinant respiratory syncytial viruscomprising the nucleic acid of claim
 1. 36-38. (Canceled)
 39. Animmunogenic composition comprising an immunologically effective amountof the recombinant respiratory syncytial virus of claim
 35. 40-45.(Canceled)
 46. An isolated or recombinant polypeptide comprising anamino acid sequence selected from the group consisting of: (a) an aminoacid sequence selected from the group consisting of SEQ ID NOs:2-11; (b)a unique amino acid subsequence comprising at least 7 contiguous aminoacid residues of any one of SEQ ID NOs:2-11; (c) an amino acid sequenceor subsequence corresponding to an artificial conservative variation ofan amino acid sequence or subsequence of (a) or (b); (d) an amino acidsequence that is greater than 99.3% identical to SEQ ID NO:2, an aminoacid sequence that is greater than 98.4% identical to SEQ ID NO:3, anamino acid sequence that is greater than 99.7% identical to SEQ ID NO:4,an amino acid sequence that is greater than 98.3% identical to SEQ IDNO:5, an amino acid sequence that is greater than 99.6% identical to SEQID NO:6, an amino acid sequence that is greater than 97.0% identical toSEQ ID NO:7, an amino acid sequence that is greater than 99.3% identicalto SEQ ID NO:8, an amino acid sequence that is greater than 99.5%identical to SEQ ID NO:9, an amino acid sequence that is greater than96.4% identical to SEQ ID NO:10, or an amino acid sequence that isgreater than 99.2% identical to SEQ ID NO:11, as determined by BLASTPusing default parameters; and, (e) an amino acid sequence or subsequencethat is specifically bound by an antibody that specifically binds to anamino acid sequence or subsequence encoded by SEQ ID NO:1, wherein saidantibody does not specifically bind to an amino acid sequence orsubsequence encoded by SEQ ID NO:13 or SEQ ID NO:14.
 47. The polypeptideof claim 46, wherein the amino acid sequence of (d) is at least 99.5%identical to SEQ ID NO:2, at least 98.6% identical to SEQ ID NO:3, atleast 99.9% identical to SEQ ID NO:4, at least 98.5% identical to SEQ IDNO:5, at least 99.8% identical to SEQ ID NO:6, at least 97.2% identicalto SEQ ID NO:7, at least 99.5% identical to SEQ ID NO:8, at least 99.7%identical to SEQ ID NO:9, at least 96.6% identical to SEQ ID NO:10, orat least 99.4% identical to SEQ ID NO:11, as determined by BLASTP usingdefault parameters.
 48. The polypeptide of claim 46, comprising at leastone artificially altered amino acid, or comprising at least oneartificially altered amino acid which comprises one or more of: adeleted amino acid, an inserted amino acid, or a substituted amino acid.49-52. (Canceled)
 53. An immunogenic composition comprising animmunologically effective amount of the polypeptide of claim
 46. 54-59.(Canceled)
 60. An isolated or recombinant polypeptide comprising theamino acid sequence of SEQ ID NO:12 with a deletion of residues 164-197,or an artificial conservative variation thereof. 61-65. (Canceled) 66.The polypeptide of claim 46, comprising the amino acid sequence of SEQID NO:8.